Generation of plants with altered protein, fiber, or oil content

ABSTRACT

The present disclosure is directed to plants that display an improved oil quantity phenotype or an improved meal quality phenotype due to altered expression of an IMQ nucleic acid. The disclosure is further directed to methods of generating plants with an improved oil quantity phenotype or improved meal quality phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. patent application Ser. No.13/941,367, filed Jul. 12, 2013; which is a divisional of U.S. patentapplication Ser. No. 13/476,801, filed May 21, 2012, now U.S. Pat. No.8,519,224, issued Aug. 27, 2013; which is a divisional of U.S. patentapplication Ser. No. 13/217,169, filed Aug. 24, 2011, now U.S. Pat. No.8,217,224, issued Jul. 10, 2012; which is a divisional of U.S. patentapplication Ser. No. 11/940,269, filed Nov. 14, 2007, now U.S. Pat. No.8,030,541, issued Oct. 4, 2011; which claims the benefit of U.S.Provisional Application No. 60/866,055, filed Nov. 15, 2006; each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure is related to transgenic plants with altered oil,protein, and/or fiber content, as well as methods of making plantshaving altered oil, protein, and/or fiber content and producing oil fromsuch plants.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Feb. 12, 2016,and is 269,350 bytes, which is incorporated by reference herein.

BACKGROUND

The ability to manipulate the composition of crop seeds, particularlythe content and composition of seed oil and protein, as well as theavailable metabolizable energy (“AME”) in the seed meal in livestock,has important applications in the agricultural industries, relating bothto processed food oils and to animal feeds. Seeds of agricultural cropscontain a variety of valuable constituents, including oil, protein andstarch. Industrial processing can separate some or all of theseconstituents for individual sale in specific applications. For instance,nearly 60% of the U.S. soybean crop is crushed by the soy processingindustry. Soy processing yields purified oil, which is sold at highvalue, while the remaining seed meal is sold for livestock feed (U.S.Soybean Board, 2001 Soy Stats). Canola seed is also crushed to produceoil and the co-product canola meal (Canola Council of Canada). Canolameal contains a high percentage of protein and a good balance of aminoacids but because it has a high fiber and phytate content, it is notreadily digested by livestock (Slominski, B. A., et al., 1999Proceedings of the 10^(th) International Rapeseed Congress, Canberra,Australia) and has a lower value than soybean meal.

Over 55% of the corn produced in the U.S. is used as animal feed (IowaCorn Growers Association). The value of the corn is directly related toits ability to be digested by livestock. Thus, it is desirable tomaximize both oil content of seeds and the AME of meal. For processedoilseeds such as soy and canola, increasing the absolute oil content ofthe seed will increase the value of such grains, while increasing theAME of meal will increase its value. For processed corn, either anincrease or a decrease in oil content may be desired, depending on howthe other major constituents are to be used. Decreasing oil may improvethe quality of isolated starch by reducing undesired flavors associatedwith oil oxidation. Alternatively, when the starch is used for ethanolproduction, where flavor is unimportant, increasing oil content mayincrease overall value.

In many feed grains, such as corn and wheat, it is desirable to increaseseed oil content, because oil has higher energy content than other seedconstituents such as carbohydrate. Oilseed processing, like most grainprocessing businesses, is a capital-intensive business; thus smallshifts in the distribution of products from the low valued components tothe high value oil component can have substantial economic impacts forgrain processors. In addition, increasing the AME of meal by adjustingseed protein and fiber content and composition, without decreasing seedoil content, can increase the value of animal feed.

Biotechnological manipulation of oils has been shown to providecompositional alteration and improvement of oil yield. Compositionalalterations include high oleic acid soybean and corn oil (U.S. Pat. Nos.6,229,033 and 6,248,939), and laurate-containing seeds (U.S. Pat. No.5,639,790), among others. Work in compositional alteration haspredominantly focused on processed oilseeds, but has been readilyextendable to non-oilseed crops, including corn. While there isconsiderable interest in increasing oil content, the only currentlypracticed biotechnology in this area is High-Oil Corn (HOC) technology(DuPont, U.S. Pat. No. 5,704,160). HOC employs high oil pollinatorsdeveloped by classical selection breeding along with elite(male-sterile) hybrid females in a production system referred to asTopCross. The TopCross High Oil system raises harvested grain oilcontent in maize from about 3.5% to about 7%, improving the energycontent of the grain.

While it has been fruitful, the HOC production system has inherentlimitations. First, the system of having a low percentage of pollinatorsresponsible for an entire field's seed set contains inherent risks,particularly in drought years. Second, oil content in current HOC fieldshas plateaued at about 9% oil. Finally, high-oil corn is not primarily abiochemical change, but rather an anatomical mutant (increased embryosize) that has the indirect result of increasing oil content. For thesereasons, an alternative high oil strategy, particularly one that derivesfrom an altered biochemical output, would be especially valuable.

Manipulation of seed composition has identified several components thatimprove the nutritive quality, digestibility, and AME in seed meal.Increasing the lysine content in canola and soybean (Falco et al., 1995Bio/Technology 13:577-582) increases the availability of this essentialamino acid and decreases the need for nutritional supplements. Soybeanvarieties with increased seed protein were shown to contain considerablymore metabolizable energy than conventional varieties (Edwards et al.,1999, Poultry Sci. 79:525-527). Decreasing the phytate content of cornseed has been shown to increase the bioavailability of amino acids inanimal feeds (Douglas et al., 2000, Poultry Sci. 79:1586-1591) anddecreasing oligosaccharide content in soybean meal increases themetabolizable energy in the meal (Parsons et al., 2000, Poultry Sci.79:1127-1131).

Soybean and canola are the most obvious target crops for the processedoil and seed meal markets since both crops are crushed for oil and theremaining meal sold for animal feed. A large body of commercial work(e.g., U.S. Pat. No. 5,952,544; PCT Application No. WO9411516)demonstrates that Arabidopsis is an excellent model for oil metabolismin these crops. Biochemical screens of seed oil composition haveidentified Arabidopsis genes for many critical biosynthetic enzymes andhave led to identification of agronomically important gene orthologs.For instance, screens using chemically mutagenized populations haveidentified lipid mutants whose seeds display altered fatty acidcomposition (Lemieux et al., 1990, Theor. Appl. Genet. 80, 234-240;James and Dooner, 1990, Theor. Appl. Genet. 80, 241-245). T-DNAmutagenesis screens (Feldmann et al., 1989, Science 243: 1351-1354) thatdetected altered fatty acid composition identified the omega 3desaturase (FADS) and delta-12 desaturase (FAD2) genes (U.S. Pat. No.5,952,544; Yadav et al., 1993, Plant Physiol. 103, 467-476; Okuley etal., 1994, Plant Cell 6(1):147-158). A screen which focused on oilcontent rather than oil quality, analyzed chemically-induced mutants forwrinkled seeds or altered seed density, from which altered seed oilcontent was inferred (Focks and Benning, 1998, Plant Physiol.118:91-101).

Another screen, designed to identify enzymes involved in production ofvery long chain fatty acids, identified a mutation in the gene encodinga diacylglycerol acyltransferase (DGAT) as being responsible for reducedtriacyl glycerol accumulation in seeds (Katavic V et al., 1995, PlantPhysiol. 108(1):399-409). It was further shown that seed-specificover-expression of the DGAT cDNA was associated with increased seed oilcontent (Jako et al., 2001, Plant Physiol. 126(2):861-74). Arabidopsisis also a model for understanding the accumulation of seed componentsthat affect meal quality. For example, Arabidopsis contains albumin andglobulin seed storage proteins found in many dicotyledonous plantsincluding canola and soybean (Shewry 1995, Plant Cell 7:945-956). Thebiochemical pathways for synthesizing components of fiber, such ascellulose and lignin, are conserved within the vascular plants, andmutants of Arabidopsis affecting these components have been isolated(reviewed in Chapel and Carpita 1998, Current Opinion in Plant Biology1:179-185).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi et al., 1992, Science 258: 1350-1353; Weigel D et al., 2000,Plant Physiology, 122:1003-1013). The inserted construct provides amolecular tag for rapid identification of the native plant whosemis-expression causes the mutant phenotype. Activation tagging may alsocause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson et al., 1996,Plant Cell 8: 659-671; Schaffer et al., 1998, Cell 93: 1219-1229;Fridborg et al., 1999, Plant Cell 11: 1019-1032; Kardailsky et al.,1999, Science 286: 1962-1965; and Christensen S et al., 1998, 9^(th)International Conference on Arabidopsis Research, Univ. ofWisconsin-Madison, June 24-28, Abstract 165).

SUMMARY

Provided herein are transgenic plants having an Improved Seed Qualityphenotype. Transgenic plants with an Improved Seed Quality phenotype mayinclude an improved oil quantity and/or an improved meal quality.Transgenic plants with improved meal quality have an Improved MealQuality (IMQ) phenotype and transgenic plants with improved oil quantityhave an Improved Oil Quantity (IOQ) phenotype. The IMQ phenotype in atransgenic plant may include altered protein and/or fiber content in anypart of the transgenic plant, for example in the seeds. The IOQphenotype in a transgenic plant may include altered oil content in anypart of the transgenic plant, for example in the seeds. In particularembodiments, a transgenic plant may include an IOQ phenotype and/or anIMQ phenotype. In some embodiments of a transgenic plant, the IMQphenotype may be an increase in protein content in the seed and/or adecrease in the fiber content of the seed. In other embodiments of atransgenic plant, the IOQ phenotype is an increase in the oil content ofthe seed (a high oil phenotype). Also provided is seed meal derived fromthe seeds of transgenic plants, wherein the seeds have altered proteincontent and/or altered fiber content. Further provided is oil derivedfrom the seeds of transgenic plants, wherein the seeds have altered oilcontent. Any of these changes can lead to an increase in the AME fromthe seed or seed meal from transgenic plants, relative to control,non-transgenic, or wild-type plants. Also provided herein is meal, feed,or food produced from any part of the transgenic plant with an IMQphenotype and/or IOQ phenotype.

In certain embodiments, the disclosed transgenic plants comprise atransformation vector comprising an IMQ nucleotide sequence that encodesor is complementary to a sequence that encodes an “IMQ” polypeptide. Inparticular embodiments, expression of an IMQ polypeptide in a transgenicplant causes an altered oil content, an altered protein content, and/oran altered fiber content in the transgenic plant. In preferredembodiments, the transgenic plant is selected from the group consistingof plants of the Brassica species, including canola and rapeseed, soy,corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor, peanut, wheat, oat and rice. Also provided is a method ofproducing oil or seed meal, comprising growing the transgenic plant andrecovering oil and/or seed meal from said plant. The disclosure furtherprovides feed, meal, grain, or seed comprising a nucleic acid sequencethat encodes an IMQ polypeptide. The disclosure also provides feed,meal, grain, or seed comprising the IMQ polypeptide, or an orthologthereof.

Examples of the disclosed transgenic plant are produced by a method thatcomprises introducing into progenitor cells of the plant a planttransformation vector comprising an IMQ nucleotide sequence thatencodes, or is complementary to a sequence that encodes, an IMQpolypeptide, and growing the transformed progenitor cells to produce atransgenic plant, wherein the IMQ polynucleotide sequence is expressed,causing an IOQ phenotype and/or and IMQ phenotype in the transgenicplant. In some specific, non-limiting examples, the method producestransgenic plants wherein expression of the IMQ polypeptide causes ahigh (increased) oil, high (increased) protein, and/or low (decreased)fiber phenotype in the transgenic plant, relative to control,non-transgenic, or wild-type plants.

Additional methods are disclosed herein of generating a plant having anIMQ and/or an IOQ phenotype, wherein a plant is identified that has anallele in its IMQ nucleic acid sequence that results in an IMQ phenotypeand/or an IOQ phenotype, compared to plants lacking the allele. Theplant can generate progeny, wherein the progeny inherit the allele andhave an IMQ phenotype and/or an IOQ phenotype. In some embodiments ofthe method, the method employs candidate gene/QTL methodology or TILLINGmethodology.

Also provided herein is a transgenic plant cell having an IMQ phenotypeand/or an IOQ phenotype. The transgenic plant cell comprises atransformation vector comprising an IMQ nucleotide sequence that encodesor is complementary to a sequence that encodes an IMQ polypeptide. Inpreferred embodiments, the transgenic plant cell is selected from thegroup consisting of plants of the Brassica species, including canola andrapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm,coconut palm, flax, castor, peanut, wheat, oat and rice. In otherembodiments, the plant cell is a seed, pollen, propagule, or embryocell. The disclosure also provides plant cells from a plant that is thedirect progeny or the indirect progeny of a plant grown from saidprogenitor cells.

DETAILED DESCRIPTION Terms

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present disclosure. Practitioners are particularly directed toSambrook et al. (Molecular Cloning: A Laboratory Manual (SecondEdition), Cold Spring Harbor Press, Plainview, N. Y., 1989) and AusubelF M et al. (Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., 1993) for definitions and terms of the art. It is to beunderstood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

As used herein, the term “IMQ phenotype” refers to plants, or any partof a plant (for example, seeds, or meal produced from seeds), with analtered protein and/or fiber content (phenotype). As provided herein,altered protein and/or fiber content includes either an increased ordecreased level of protein and/or fiber content in plants, seeds or seedmeal. Any combination of these changes can lead to an IMQ phenotype. Forexample, in one specific non-limiting example, an IMQ phenotype canrefer to increased protein and decreased fiber content. In anotherspecific non-limiting example, an IMQ phenotype can refer to unchangedprotein and decreased fiber content. In yet another specificnon-limiting example, an IMQ phenotype can refer to increased proteinand unchanged fiber content. It is also provided that any combination ofthese changes can lead to an increase in the AME (availablemetabolizable energy) from the seed or meal generated from the seed. AnIMQ phenotype also includes an improved seed quality (ISQ) phenotype oran improved seed meal quality phenotype.

As used herein, the term “IOQ phenotype” refers to plants, or any partof a plant (for example, seeds), with an altered oil content(phenotype). As provided herein, altered oil content includes anincreased, for example a high, oil content in plants or seeds. In someembodiments, a transgenic plant can express both an IOQ phenotype and anIMQ phenotype. In specific, non-limiting examples, a transgenic planthaving a combination of an IOQ phenotype and an IMQ phenotype can leadto an increase in the AME (available metabolizable energy) from the seedor meal generated from the seed. An IOQ phenotype also includes animproved seed quality (ISQ) phenotype.

As used herein, the term “available metabolizable energy” (AME) refersto the amount of energy in the feed that is able to be extracted bydigestion in an animal and is correlated with the amount of digestibleprotein and oil available in animal meal. AME is determined byestimating the amount of energy in the feed prior to feeding andmeasuring the amount of energy in the excreta of the animal followingconsumption of the feed. In one specific, non-limiting example, atransgenic plant with an increase in AME includes transgenic plants withaltered seed protein and/or fiber content and without a decrease in seedoil content (seed oil content remains unchanged or is increased),resulting in an increase in the value of animal feed derived from theseed.

As used herein, the term “content” refers to the type and relativeamount of, for instance, a seed or seed meal component.

As used herein, the term “fiber” refers to non-digestible components ofthe plant seed including cellular components such as cellulose,hemicellulose, pectin, lignin, and phenolics.

As used herein, the term “meal” refers to seed components remainingfollowing the extraction of oil from the seed. Examples of components ofmeal include protein and fiber.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from, a controlsequence/DNA coding sequence combination found in the native plant.Specific, non-limiting examples of a heterologous nucleic acid sequenceinclude an IMQ nucleic acid sequence, or a fragment, derivative(variant), or ortholog thereof.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequences.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-altered or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, includes “transfection,” “transformation,” and“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus), as well asfrom plant seeds, pollen, propagules, and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature. In oneembodiment, a wild-type plant is also a control plant. In anotherembodiment, a wild-type plant is a non-transgenic plant.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant (for example, a transgenicplant with any combination of an altered oil content, an altered proteincontent, and/or an altered fiber content) in any part of the transgenicplant, for example the seeds, relative to a similar non-transgenicplant. As used herein, the term “altered” refers to either an increaseor a decrease of a plant trait or phenotype (for example, oil content,protein content, and/or fiber content) in a transgenic plant, relativeto a similar non-transgenic plant. In one specific, non-limitingexample, a transgenic plant with a modified trait includes a plant withan increased oil content, increased protein content, and/or decreasedfiber content relative to a similar non-transgenic plant. In anotherspecific, non-limiting example, a transgenic plant with a modified traitincludes unchanged oil content, increased protein content, and/ordecreased fiber content relative to a similar non-transgenic plant. Inyet another specific, non-limiting example, a transgenic plant with amodified trait includes an increased oil content, increased proteincontent, and/or unchanged fiber content relative to a similarnon-transgenic plant. Specific, non-limiting examples of a change inphenotype include an IMQ phenotype or an IOQ phenotype.

An “interesting phenotype (trait)” with reference to a transgenic plantrefers to an observable or measurable phenotype demonstrated by a T1and/or subsequent generation plant, which is not displayed by thecorresponding non-transgenic plant (i.e., a genotypically similar plantthat has been raised or assayed under similar conditions). Aninteresting phenotype may represent an improvement in the plant (forexample, increased oil content, increased protein content, and/ordecreased fiber content in seeds of the plant) or may provide a means toproduce improvements in other plants. An “improvement” is a feature thatmay enhance the utility of a plant species or variety by providing theplant with a unique and/or novel phenotype or quality. Such transgenicplants may have an improved phenotype, such as an IMQ phenotype or anIOQ phenotype.

The phrase “altered oil content phenotype” refers to a measurablephenotype of a genetically modified (transgenic) plant, where the plantdisplays a statistically significant increase or decrease in overall oilcontent (i.e., the percentage of seed mass that is oil), as compared tothe similar, but non-modified (non-transgenic) plant. A high oilphenotype refers to an increase in overall oil content. The phrase“altered protein content phenotype” refers to measurable phenotype of agenetically modified plant, where the plant displays a statisticallysignificant increase or decrease in overall protein content (i.e., thepercentage of seed mass that is protein), as compared to the similar,but non-modified plant. A high protein phenotype refers to an increasein overall protein content. The phrase “altered fiber content phenotype”refers to measurable phenotype of a genetically modified plant, wherethe plant displays a statistically significant increase or decrease inoverall fiber content (i.e., the percentage of seed mass that is fiber),as compared to the similar, but non-modified plant. A low fiberphenotype refers to decrease in overall fiber content.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild-type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified or altered plant phenotype or trait. Relative to a plant orplant line, the term “mutant” refers to a plant or plant line which hasa modified or altered plant phenotype or trait, where the modified oraltered phenotype or trait is associated with the modified or alteredexpression of a wild-type polynucleotide sequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic. T3 plants are generated from T2 plants,etc. As used herein, the “direct progeny” of a given plant derives fromthe seed (or, sometimes, other tissue) of that plant and is in theimmediately subsequent generation; for instance, for a given lineage, aT2 plant is the direct progeny of a T1 plant. The “indirect progeny” ofa given plant derives from the seed (or other tissue) of the directprogeny of that plant, or from the seed (or other tissue) of subsequentgenerations in that lineage; for instance, a T3 plant is the indirectprogeny of a T1 plant.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. Providedherein is a transgenic plant cell having an IMQ phenotype and/or an IOQphenotype. The transgenic plant cell comprises a transformation vectorcomprising an IMQ nucleotide sequence that encodes or is complementaryto a sequence that encodes an IMQ polypeptide. In preferred embodiments,the transgenic plant cell is selected from the group consisting ofplants of the Brassica species, including canola and rapeseed, soy,corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor, peanut, wheat, oat and rice. In other embodiments, the plantcell is a seed, pollen, propagule, or embryo cell. The disclosure alsoprovides plant cells from a plant that is the direct progeny or theindirect progeny of a plant grown from said progenitor cells. The classof plants which can be used in the methods of the present disclosure isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants.

As used herein, “transgenic plant” includes a plant that compriseswithin its genome a heterologous polynucleotide. The heterologouspolynucleotide can be either stably integrated into the genome, or canbe extra-chromosomal. Preferably, the polynucleotide of the presentdisclosure is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed,” “transfected,” or“transgenic.” Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Disclosed herein are transgenic plants having an Improved Seed Qualityphenotype. Transgenic plants with an Improved Seed Quality phenotype mayinclude an improved oil quantity and/or an improved meal quality.Transgenic plants with improved meal quality have an IMQ phenotype andtransgenic plants with improved oil quantity have an IOQ phenotype. TheIMQ phenotype in a transgenic plant may include altered protein and/orfiber content in any part of the transgenic plant, for example in theseeds. The IOQ phenotype in a transgenic plant may include altered oilcontent in any part of the transgenic plant, for example in the seeds.In particular embodiments, a transgenic plant may include an IOQphenotype and/or an IMQ phenotype. In some embodiments of a transgenicplant, the IMQ phenotype may be an increase in protein content in theseed and/or a decrease in the fiber content of the seed. In otherembodiments of a transgenic plant, the IOQ phenotype is an increase inthe oil content of the seed (a high oil phenotype). Also provided isseed meal derived from the seeds of transgenic plants, wherein the seedshave altered protein content and/or altered fiber content. Furtherprovided is oil derived from the seeds of transgenic plants, wherein theseeds have altered oil content. Any of these changes can lead to anincrease in the AME from the seed or seed meal from transgenic plants,relative to control, non-transgenic, or wild-type plants. Also providedherein is meal, feed, or food produced from any part of the transgenicplant with an IMQ phenotype and/or IOQ phenotype.

In certain embodiments, the disclosed transgenic plants comprise atransformation vector comprising an IMQ nucleotide sequence that encodesor is complementary to a sequence that encodes an “IMQ” polypeptide. Inparticular embodiments, expression of an IMQ polypeptide in a transgenicplant causes an altered oil content, an altered protein content, and/oran altered fiber content in the transgenic plant. In preferredembodiments, the transgenic plant is selected from the group consistingof plants of the Brassica species, including canola and rapeseed, soy,corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor, peanut, wheat, oat and rice. Also provided is a method ofproducing oil or seed meal, comprising growing the transgenic plant andrecovering oil and/or seed meal from said plant. The disclosure furtherprovides feed, meal, grain, or seed comprising a nucleic acid sequencethat encodes an IMQ polypeptide. The disclosure also provides feed,meal, grain, or seed comprising the IMQ polypeptide, or an orthologthereof.

Various methods for the introduction of a desired polynucleotidesequence encoding the desired protein into plant cells are available andknown to those of skill in the art and include, but are not limited to:(1) physical methods such as microinjection, electroporation, andmicroprojectile mediated delivery (biolistics or gene gun technology);(2) virus mediated delivery methods; and (3) Agrobacterium-mediatedtransformation methods (see, for example, WO 2007/053482 and WO2005/107437, which are incorporated herein by reference in theirentirety).

The most commonly used methods for transformation of plant cells are theAgrobacterium-mediated DNA transfer process and the biolistics ormicroprojectile bombardment mediated process (i.e., the gene gun).Typically, nuclear transformation is desired but where it is desirableto specifically transform plastids, such as chloroplasts or amyloplasts,plant plastids may be transformed utilizing a microprojectile-mediateddelivery of the desired polynucleotide.

Agrobacterium-mediated transformation is achieved through the use of agenetically engineered soil bacterium belonging to the genusAgrobacterium. A number of wild-type and disarmed strains ofAgrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti orRi plasmids can be used for gene transfer into plants. Gene transfer isdone via the transfer of a specific DNA known as “T-DNA” that can begenetically engineered to carry any desired piece of DNA into many plantspecies.

Agrobacterium-mediated genetic transformation of plants involves severalsteps. The first step, in which the virulent Agrobacterium and plantcells are first brought into contact with each other, is generallycalled “inoculation.” Following the inoculation, the Agrobacterium andplant cells/tissues are permitted to be grown together for a period ofseveral hours to several days or more under conditions suitable forgrowth and T-DNA transfer. This step is termed “co-culture.” Followingco-culture and T-DNA delivery, the plant cells are treated withbactericidal or bacteriostatic agents to kill the Agrobacteriumremaining in contact with the explant and/or in the vessel containingthe explant. If this is done in the absence of any selective agents topromote preferential growth of transgenic versus non-transgenic plantcells, then this is typically referred to as the “delay” step. If donein the presence of selective pressure favoring transgenic plant cells,then it is referred to as a “selection” step. When a “delay” is used, itis typically followed by one or more “selection” steps.

With respect to microprojectile bombardment (U.S. Pat. No. 5,550,318;U.S. Pat. No. 5,538,880, U.S. Pat. No. 5,610,042; and PCT Publication WO95/06128; each of which is specifically incorporated herein by referencein its entirety), particles are coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, platinum, and preferably, gold.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species thathave been transformed by microprojectile bombardment include monocotspecies such as maize (PCT Publication No. WO 95/06128), barley, wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference in itsentirety), rice, oat, rye, sugarcane, and sorghum, as well as a numberof dicots including tobacco, soybean (U.S. Pat. No. 5,322,783,incorporated herein by reference in its entirety), sunflower, peanut,cotton, tomato, and legumes in general (U.S. Pat. No. 5,563,055,incorporated herein by reference in its entirety).

To select or score for transformed plant cells regardless oftransformation methodology, the DNA introduced into the cell contains agene that functions in a regenerable plant tissue to produce a compoundthat confers upon the plant tissue resistance to an otherwise toxiccompound. Genes of interest for use as a selectable, screenable, orscorable marker would include but are not limited to GUS, greenfluorescent protein (GFP), luciferase (LUX), antibiotic or herbicidetolerance genes. Examples of antibiotic resistance genes include thepenicillins, kanamycin (and neomycin, G418, bleomycin), methotrexate(and trimethoprim), chloramphenicol, and tetracycline. Polynucleotidemolecules encoding proteins involved in herbicide tolerance are known inthe art, and include, but are not limited to a polynucleotide moleculeencoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) describedin U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, and U.S. Pat. No.6,040,497 and aroA described in U.S. Pat. No. 5,094,945 for glyphosatetolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn)described in U.S. Pat. No. 4,810,648 for Bromoxynil tolerance; apolynucleotide molecule encoding phytoene desaturase (crtI) described inMisawa et al. (Plant J. 4:833-840, 1993) and Misawa et al. (Plant J.6:481-489, 1994) for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, also known as ALS) describedin Sathasiivan et al. (Nucl. Acids Res. 18:2188-2193, 1990) fortolerance to sulfonylurea herbicides; and the bar gene described inDeBlock et al. (EMBO J. 6:2513-2519, 1987) for glufosinate and bialaphostolerance.

The regeneration, development, and cultivation of plants from varioustransformed explants are well documented in the art. This regenerationand growth process typically includes the steps of selecting transformedcells and culturing those individualized cells through the usual stagesof embryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil. Cells that survive the exposure to the selectiveagent, or cells that have been scored positive in a screening assay, maybe cultured in media that supports regeneration of plants. Developingplantlets are transferred to soil less plant growth mix, and hardenedoff, prior to transfer to a greenhouse or growth chamber for maturation.

The present disclosure can be used with any transformable cell ortissue. By transformable as used herein is meant a cell or tissue thatis capable of further propagation to give rise to a plant. Those ofskill in the art recognize that a number of plant cells or tissues aretransformable in which after insertion of exogenous DNA and appropriateculture conditions the plant cells or tissues can form into adifferentiated plant. Tissue suitable for these purposes can include butis not limited to immature embryos, scutellar tissue, suspension cellcultures, immature inflorescence, shoot meristem, nodal explants, callustissue, hypocotyl tissue, cotyledons, roots, and leaves.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al.,Scientia Sinica 18:659, 1975) supplemented with additional plant growthregulators including but not limited to auxins, cytokinins, ABA, andgibberellins. Those of skill in the art are familiar with the variety oftissue culture media, which when supplemented appropriately, supportplant tissue growth and development and are suitable for planttransformation and regeneration. These tissue culture media can eitherbe purchased as a commercial preparation, or custom prepared andmodified. Those of skill in the art are aware that media and mediasupplements such as nutrients and growth regulators for use intransformation and regeneration and other culture conditions such aslight intensity during incubation, pH, and incubation temperatures thatcan be optimized for the particular variety of interest.

One of ordinary skill will appreciate that, after an expression cassetteis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

Identification of Plants with an Improved Oil Quantity Phenotype and/orImproved Meal Quality Phenotype

An Arabidopsis activation tagging screen (ACTTAG) was used to identifythe association between 1) ACTTAG plant lines with an altered protein,fiber and/or oil content (phenotype, for example, see columns 4, 5 and6, respectively, of Table 1, below) and 2) the nucleic acid sequencesidentified in column 3 of Tables 2 and 3, wherein each nucleic acidsequence is provided with a gene alias or an IMQ designation (IMQ#; seecolumn 1 in Tables 1, 2, and 3). Briefly, and as further described inthe Examples, a large number of Arabidopsis plants were mutated with thepSKI015 vector, which comprises a T-DNA from the Ti plasmid ofAgrobacterium tumefaciens, a viral enhancer element, and a selectablemarker gene (Weigel et al., 2000, Plant Physiology, 122:1003-1013). Whenthe T-DNA inserts into the genome of transformed plants, the enhancerelement can cause up-regulation of genes in the vicinity, generallywithin about nine kilobases (kb) of the enhancers. T1 plants wereexposed to the selective agent in order to specifically recovertransformed plants that expressed the selectable marker and thereforeharbored T-DNA insertions. T1 plants were allowed to grow to maturity,self-fertilize and produce seed. T2 seed was harvested, labeled andstored. To amplify the seed stocks, about eighteen T2 were sown in soiland, after germination, exposed to the selective agent to recovertransformed T2 plants. T3 seed from these plants was harvested andpooled. Oil, protein and fiber content of the seed were estimated usingNear Infrared Spectroscopy (NIR) as described in the Examples.

Quantitative determination of fatty acid (FA) content (column 7,Table 1) in T2 seeds was performed using the following methods. A sampleof 15 to 20 T2 seeds from each line tested. This sample generallycontained plants with homozygous insertions, no insertions, andhemizygous insertions in a standard 1:1:2 ratios. The seed sample wasmassed on UMT-2 ultra-microbalance (Mettler-Toledo Co., Ohio, USA) andthen transferred to a glass extraction vial. Lipids were extracted fromthe seeds and trans-esterified in 500 μl 2.5% H₂SO₄ in MeOH for 3 hoursat 80° C., following the method of Browse et al. (Biochem J 235:25-31,1986) with modifications. A known amount of heptadecanoic acid wasincluded in the reaction as an internal standard. 750 μl of water and400 μl of hexane were added to each vial, which was then shakenvigorously and allowed to phase separate. Reaction vials were loadeddirectly onto gas chromatography (GC) for analysis and the upper hexanephase was sampled by the autosampler. Gas chromatography with FlameIonization detection was used to separate and quantify the fatty acidmethyl esters. Agilent 6890 Plus GC's were used for separation withAgilent Innowax columns (30 m×0.25 mm ID, 250 um film thickness). Thecarrier gas was Hydrogen at a constant flow of 2.5 ml/minute. 1 μl ofsample was injected in splitless mode (inlet temperature 220° C., Purgeflow 15 ml/min at 1 minute). The oven was programmed for an initialtemperature of 105° C., initial time 0.5 minutes, followed by a ramp of60° C. per minute to 175° C., a 40° C./minute ramp to 260° C. with afinal hold time of 2 minutes. Detection was by Flame Ionization(Temperature 275° C., Fuel flow 30.0 ml/min, Oxidizer 400.0 ml/min).Instrument control and data collection and analysis were monitored usingthe Millennium Chromatography Management System (Version 3.2, WatersCorporation, Milford, Mass.). Peaks were initially identified bycomparison with standards. Integration and quantification were performedautomatically, but all analyses were subsequently examined manually toverify correct peak identification and acceptable signal to noise ratiobefore inclusion of the derived results in the study.

The association of an IMQ nucleic acid sequence with an IMQ phenotype oran IOQ phenotype was discovered by analysis of the genomic DNA sequenceflanking the T-DNA insertion in the ACTTAG line identified in column 3of Table 1. An ACTTAG line is a family of plants derived from a singleplant that was transformed with a T-DNA element containing four tandemcopies of the CaMV 35S enhancers. Accordingly, the disclosed IMQ nucleicacid sequences and/or polypeptides may be employed in the development oftransgenic plants having an improved seed quality phenotype, includingan IMQ phenotype and/or an IOQ phenotype. IMQ nucleic acid sequences maybe used in the generation of transgenic plants, such as oilseed crops,that provide improved oil yield from oilseed processing and result in anincrease in the quantity of oil recovered from seeds of the transgenicplant. IMQ nucleic acid sequences may also be used in the generation oftransgenic plants, such as feed grain crops, that provide an IMQphenotype resulting in increased energy for animal feeding, for example,seeds or seed meal with an altered protein and/or fiber content,resulting in an increase in AME. IMQ nucleic acid sequences may furtherbe used to increase the oil content of specialty oil crops, in order toaugment yield and/or recovery of desired unusual fatty acids. Transgenicplants that have been genetically modified to express IMQ polypeptidescan be used in the production of seeds, wherein the transgenic plantsare grown, and oil and seed meal are obtained from plant parts (e.g.seed) using standard methods.

IMQ Nucleic Acids and Polypeptides

The IMQ designation for each of the IMQ nucleic acid sequencesdiscovered in the activation tagging screen described herein are listedin column 1 of Tables 1-3, below. The disclosed IMQ polypeptides arelisted in column 5 of Table 2 and column 4 of Table 3. As used herein,the term “IMQ polypeptide” refers to any polypeptide that when expressedin a plant causes an IMQ phenotype and/or an IOQ phenotype in any partof the plant, for example the seeds. In one embodiment, an IMQpolypeptide refers to a full-length IMQ protein, or a fragment,derivative (variant), or ortholog thereof that is “functionally active,”such that the protein fragment, derivative, or ortholog exhibits one ormore or the functional activities associated with one or more of thedisclosed full-length IMQ polypeptides, for example, the amino acidsequences provided in the GenBank entry referenced in column 5 of Table2, which correspond to the amino acid sequences set forth as SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ IDNO: 98, or SEQ ID NO: 100, or an ortholog thereof. In one preferredembodiment, a functionally active IMQ polypeptide causes an IMQphenotype and/or an IOQ phenotype in a transgenic plant. In anotherembodiment, a functionally active IMQ polypeptide causes an altered oil,protein, and/or fiber content phenotype (for example, an altered seedmeal content phenotype) when mis-expressed in a plant. In otherpreferred embodiments, mis-expression of the IMQ polypeptide causes ahigh oil (such as, increased oil), high protein (such as, increasedprotein), and/or low fiber (such as, decreased fiber) phenotype in aplant. In another embodiment, mis-expression of the IMQ polypeptidecauses an improved AME of meal. In yet another embodiment, afunctionally active IMQ polypeptide can rescue defective (includingdeficient) endogenous IMQ activity when expressed in a plant or in plantcells; the rescuing polypeptide may be from the same or from a differentspecies as the species with the defective polypeptide activity. Thedisclosure also provides feed, meal, grain, food, or seed comprising theIMQ polypeptide, or a fragment, derivative (variant), or orthologthereof.

In another embodiment, a functionally active fragment of a full lengthIMQ polypeptide (for example, a functionally active fragment of a nativepolypeptide having the amino acid sequence set forth as SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88,SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO:98, or SEQ ID NO: 100, or a naturally occurring ortholog thereof)retains one or more of the biological properties associated with thefull-length IMQ polypeptide, such as signaling activity, bindingactivity, catalytic activity, or cellular or extra-cellular localizingactivity. An IMQ fragment preferably comprises an IMQ domain, such as aC- or N-terminal or catalytic domain, among others, and preferablycomprises at least 10, preferably at least 20, more preferably at least25, and most preferably at least 50 contiguous amino acids of an IMQprotein. Functional domains of IMQ genes are listed in column 8 of Table2 and can be identified using the PFAM program (Bateman A et al., 1999,Nucleic Acids Res. 27:260-262) or INTERPRO (Mulder et al., 2003, NucleicAcids Res. 31, 315-318) program. Functionally active variants offull-length IMQ polypeptides, or fragments thereof, include polypeptideswith amino acid insertions, deletions, or substitutions that retain oneof more of the biological properties associated with the full-length IMQpolypeptide. In some cases, variants are generated that change thepost-translational processing of an IMQ polypeptide. For instance,variants may have altered protein transport or protein localizationcharacteristics, or altered protein half-life, compared to the nativepolypeptide.

As used herein, the term “IMQ nucleic acid” refers to any polynucleotidethat when expressed in a plant causes an IMQ phenotype and/or an IOQphenotype in any part of the plant, for example the seeds. In oneembodiment, an IMQ polynucleotide encompasses nucleic acids with thesequence provided in or complementary to the GenBank entry referenced incolumn 3 of Table 2, which correspond to nucleic acid sequences setforth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27,SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ IDNO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ IDNO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99, as well as functionallyactive fragments, derivatives, or orthologs thereof. An IMQ nucleic acidof this disclosure may be DNA, derived from genomic DNA or cDNA, or RNA.Genomic sequences of the genes listed in Table 2 are known and availablein public databases such as GenBank.

In one embodiment, a functionally active IMQ nucleic acid encodes or iscomplementary to a nucleic acid that encodes a functionally active IMQpolypeptide. A functionally active IMQ nucleic acid also includesgenomic DNA that serves as a template for a primary RNA transcript(i.e., an mRNA precursor) that requires processing, such as splicing,before encoding the functionally active IMQ polypeptide. An IMQ nucleicacid can include other non-coding sequences, which may or may not betranscribed; such sequences include 5′ and 3′ UTRs, polyadenylationsignals and regulatory sequences that control gene expression, amongothers, as are known in the art. Some polypeptides require processingevents, such as proteolytic cleavage, covalent modification, etc., inorder to become fully active. Accordingly, functionally active nucleicacids may encode the mature or the pre-processed IMQ polypeptide, or anintermediate form. An IMQ polynucleotide can also include heterologouscoding sequences, for example, sequences that encode a marker includedto facilitate the purification of the fused polypeptide, or atransformation marker. In another embodiment, a functionally active IMQnucleic acid is capable of being used in the generation ofloss-of-function IMQ phenotypes, for instance, via antisensesuppression, co-suppression, etc. The disclosure also provides feed,meal, grain, food, or seed comprising a nucleic acid sequence thatencodes an IMQ polypeptide.

In one preferred embodiment, an IMQ nucleic acid used in the disclosedmethods comprises a nucleic acid sequence that encodes, or iscomplementary to a sequence that encodes, an IMQ polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity to a disclosed IMQ polypeptide sequence, for example the aminoacid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ IDNO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92,SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100.

In another embodiment, an IMQ polypeptide comprises a polypeptidesequence with at least 50% or 60% identity to a disclosed IMQpolypeptide sequence (for example, the amino acid sequence set forth asSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ IDNO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO:96, SEQ ID NO: 98, or SEQ ID NO: 100) and may have at least 70%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a disclosed IMQpolypeptide sequence. In a further embodiment, an IMQ polypeptidecomprises 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity to a disclosed IMQ polypeptide sequence, and may include aconserved protein domain of the IMQ polypeptide (such as the proteindomain(s) listed in column 8 of Table 2). In another embodiment, an IMQpolypeptide comprises a polypeptide sequence with at least 50%, 60%,70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to afunctionally active fragment of the polypeptide referenced in column 5of Table 2. In yet another embodiment, an IMQ polypeptide comprises apolypeptide sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%,98%, or 99% identity to the polypeptide sequence of the GenBank entryreferenced in column 5 of Table 2 over its entire length and comprises aconserved protein domain(s) listed in column 8 of Table 2.

In another aspect, an IMQ polynucleotide sequence is at least 50% to 60%identical over its entire length to a disclosed IMQ nucleic acidsequence, such as the nucleic acid sequence set forth as SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ IDNO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQ ID NO: 77, SEQID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87,SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO:97, or SEQ ID NO: 99, or nucleic acid sequences that are complementaryto such an IMQ sequence, and may comprise at least 70%, 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity to the disclosed IMQ sequence,or a functionally active fragment thereof, or complementary sequences.In another embodiment, a disclosed IMQ nucleic acid comprises a nucleicacid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:73 SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ IDNO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, or SEQ ID NO: 99, or nucleicacid sequences that are complementary to such an IMQ sequence, andnucleic acid sequences that have substantial sequence homology to a suchIMQ sequences. As used herein, the phrase “substantial sequencehomology” refers to those nucleic acid sequences that have slight orinconsequential sequence variations from such IMQ sequences, i.e., thesequences function in substantially the same manner and encode an IMQpolypeptide.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in an identifiedsequence identical with the nucleotides or amino acids in the subjectsequence (or specified portion thereof), after aligning the sequencesand introducing gaps, if necessary to achieve the maximum percentsequence identity, as generated by the program WU-BLAST-2.0a19 (Altschulet al., J. Mol. Biol., 1990, 215:403-410) with search parameters set todefault values. The HSP S and HSP S2 parameters are dynamic values andare established by the program itself depending upon the composition ofthe particular sequence and composition of the particular databaseagainst which the sequence of interest is being searched. A “percent (%)identity value” is determined by the number of matching identicalnucleotides or amino acids divided by the sequence length for which thepercent identity is being reported. “Percent (%) amino acid sequencesimilarity” is determined by performing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation. A conservative amino acid substitution is one in which anamino acid is substituted for another amino acid having similarproperties such that the folding or activity of the protein is notsignificantly affected. Aromatic amino acids that can be substituted foreach other are phenylalanine, tryptophan, and tyrosine; interchangeablehydrophobic amino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that selectively hybridize to the disclosed IMQnucleic acid sequences (for example, the nucleic acid sequence set forthas SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, or SEQ ID NO: 99). The stringency of hybridizationcan be controlled by temperature, ionic strength, pH, and the presenceof denaturing agents such as formamide during hybridization and washing.Conditions routinely used are well known (see, e.g., Current Protocol inMolecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers(1994); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual(Second Edition), Cold Spring Harbor Press, Plainview, N.Y.,).

In some embodiments, a nucleic acid molecule of the disclosure iscapable of hybridizing to a nucleic acid molecule containing thedisclosed nucleotide sequence under stringent hybridization conditionsthat are: prehybridization of filters containing nucleic acid for 8hours to overnight at 65° C. in a solution comprising 6× single strengthcitrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0),5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herringsperm DNA; hybridization for 18-20 hours at 65° C. in a solutioncontaining 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05%sodium pyrophosphate; and washing of filters at 65° C. for 1 h in asolution containing 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate). Inother embodiments, moderately stringent hybridization conditions areused that are: pretreatment of filters containing nucleic acid for 6 hat 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl(pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/mldenatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in asolution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively, lowstringency conditions can be used that comprise: incubation for 8 hoursto overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridizationin the same buffer for 18 to 20 hours; and washing of filters in 1×SSCat about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding an IMQ polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura et al., 1999, Nucleic Acids Res. 27:292).Such sequence variants may be used in the methods disclosed herein.

The disclosed methods may use orthologs of a disclosed Arabidopsis IMQnucleic acid sequence. Representative putative orthologs of each of thedisclosed Arabidopsis IMQ genes are identified in column 3 of Table 3,below. Methods of identifying the orthologs in other plant species areknown in the art. In general, orthologs in different species retain thesame function, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, 1998, Proc. Natl. Acad. Sci., 95:5849-5856;Huynen M A et al., 2000, Genome Research, 10:1204-1210).

Programs for multiple sequence alignment, such as CLUSTAL (Thompson J Det al., 1994, Nucleic Acids Res. 22:4673-4680) may be used to highlightconserved regions and/or residues of orthologous proteins and togenerate phylogenetic trees. In a phylogenetic tree representingmultiple homologous sequences from diverse species (e.g., retrievedthrough BLAST analysis), orthologous sequences from two speciesgenerally appear closest on the tree with respect to all other sequencesfrom these two species. Structural threading or other analysis ofprotein folding (e.g., using software by ProCeryon, Biosciences,Salzburg, Austria) may also identify potential orthologs. Nucleic acidhybridization methods may also be used to find orthologous genes and arepreferred when sequence data are not available. Degenerate PCR andscreening of cDNA or genomic DNA libraries are common methods forfinding related gene sequences and are well known in the art (see, e.g.,Sambrook, 1989, Molecular Cloning: A Laboratory Manual (Second Edition),Cold Spring Harbor Press, Plainview, N.Y.; Dieffenbach and Dveksler,1995, PCR Primer: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY). For instance, methods for generating a cDNA library from theplant species of interest and probing the library with partiallyhomologous gene probes are described in Sambrook et al. A highlyconserved portion of the Arabidopsis IMQ coding sequence may be used asa probe. IMQ ortholog nucleic acids may hybridize to the nucleic acid ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, or SEQ ID NO: 99 under high, moderate, or lowstringency conditions. After amplification or isolation of a segment ofa putative ortholog, that segment may be cloned and sequenced bystandard techniques and utilized as a probe to isolate a complete cDNAor genomic DNA clone.

Alternatively, it is possible to initiate an EST project to generate adatabase of sequence information for the plant species of interest. Inanother approach, antibodies that specifically bind known IMQpolypeptides are used for ortholog isolation (see, e.g., Harlow andLane, 1988, 1999, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York). Western blot analysis can determine that anIMQ ortholog (i.e., a protein orthologous to a disclosed IMQpolypeptide) is present in a crude extract of a particular plantspecies. When reactivity is observed, the sequence encoding thecandidate ortholog may be isolated by screening expression librariesrepresenting the particular plant species. Expression libraries can beconstructed in a variety of commercially available vectors, includinglambda gt11, as described in Sambrook, et al., 1989. Once the candidateortholog(s) are identified by any of these means, candidate orthologoussequence are used as bait (the “query”) for the reverse BLAST againstsequences from Arabidopsis or other species in which IMQ nucleic acidand/or polypeptide sequences have been identified.

IMQ nucleic acids and polypeptides may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR), as previously described, are well known in theart. Alternatively, nucleic acid sequence may be synthesized. Any knownmethod, such as site directed mutagenesis (Kunkel T A et al., 1991,Methods Enzymol. 204:125-39), may be used to introduce desired changesinto a cloned nucleic acid.

In general, the methods disclosed herein involve incorporating thedesired form of the IMQ nucleic acid into a plant expression vector fortransformation of plant cells, and the IMQ polypeptide is expressed inthe host plant. Transformed plants and plant cells expressing an IMQpolypeptide express an IMQ phenotype and/or an IOQ phenotype and, in onespecific, non-limiting example, may have high (increased) oil, high(increased) protein, and/or low (decreased) fiber content.

An “isolated” IMQ nucleic acid molecule is other than in the form orsetting in which it is found in nature, and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the IMQ nucleic acid.However, an isolated IMQ nucleic acid molecule includes IMQ nucleic acidmolecules contained in cells that ordinarily express IMQ where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

Generation of Genetically Modified Plants with an Improved Oil QuantityPhenotype and/or an Improved Meal Quality Phenotype

The disclosed IMQ nucleic acids and polypeptides may be used in thegeneration of transgenic plants having a modified or altered oil,protein, and/or fiber content phenotype. As used herein, an “altered oilcontent (phenotype)” may refer to altered oil content in any part of theplant. In a preferred embodiment, altered expression of the IMQ gene ina plant is used to generate plants with a high oil content (phenotype).As used herein, an “altered protein content (phenotype)” may refer toaltered protein content in any part of the plant. In a preferredembodiment, altered expression of the IMQ gene in a plant is used togenerate plants with a high (or increased) protein content (phenotype).As used herein, an “altered fiber content (phenotype)” may refer toaltered fiber content in any part of the plant. In a preferredembodiment, altered expression of the IMQ gene in a plant is used togenerate plants with a low (or decreased) fiber content (phenotype). Thealtered oil, protein, and/or fiber content is often observed in seeds.Examples of a transgenic plant include plants comprising a planttransformation vector with a nucleotide sequence that encodes or iscomplementary to a sequence that encodes an IMQ polypeptide having theamino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ IDNO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ IDNO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, or SEQ ID NO: 100,or an ortholog thereof.

Transgenic plants, such as corn, soybean and canola containing thedisclosed nucleic acid sequences, can be used in the production ofvegetable oil and meal. Vegetable oil is used in a variety of foodproducts, while meal from seed is used as an animal feed. Afterharvesting seed from transgenic plants, the seed is cleaned to removeplant stalks and other material and then flaked in roller mills to breakthe hulls. The crushed seed is heated to 75-100° C. to denaturehydrolytic enzymes, lyse the unbroken oil containing cells, and allowsmall oil droplets to coalesce. Most of the oil is then removed (and canbe recovered) by pressing the seed material in a screw press. Theremaining oil is removed from the presscake by extraction with andorganic solvents, such as hexane. The solvent is removed from the mealby heating it to approximately 100° C. After drying, the meal is thengranulated to a consistent form. The meal, containing the protein,digestible carbohydrate, and fiber of the seed, may be mixed with othermaterials prior to being used as an animal feed.

The methods described herein for generating transgenic plants aregenerally applicable to all plants. Although activation tagging and geneidentification is carried out in Arabidopsis, the IMQ nucleic acidsequence (or an ortholog, variant or fragment thereof) may be expressedin any type of plant. In a preferred embodiment, oil-producing plantsproduce and store triacylglycerol in specific organs, primarily inseeds. Such species include soybean (Glycine max), rapeseed and canola(including Brassica napus, B. campestris), sunflower (Helianthus annus),cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao),safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconutpalm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinuscommunis), and peanut (Arachis hypogaea), as well as wheat, rice andoat. Fruit- and vegetable-bearing plants, grain-producing plants,nut-producing plants, rapid cycling Brassica species, alfalfa (Medicagosativa), tobacco (Nicotiana), turfgrass (Poaceae family), other foragecrops, and wild species may also be a source of unique fatty acids. Inother embodiments, any plant expressing the IMQ nucleic acid sequencecan also express increased protein and/or decreased fiber content in aspecific plant part or organ, such as in seeds.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present disclosure. For example, theconstructs can be introduced in a variety of forms including, but notlimited to, as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to, Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment, calcium-phosphate-DNAco-precipitation, or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising an IMQ polynucleotide mayencode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.). A construct orvector may include a plant promoter to express the nucleic acid moleculeof choice. In a preferred embodiment, the promoter is a plant promoter.

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature. Of particular relevance are methodsto transform commercially important crops, such as plants of theBrassica species, including canola and rapeseed, (De Block et al., 1989,Plant Physiol., 91:694-701), sunflower (Everett et al., 1987,Bio/Technology, 5:1201), soybean (Christou et al., 1989, Proc. Natl.Acad. Sci USA, 86:7500-7504; Kline et al., 1987, Nature, 327:70), wheat,rice and oat.

Expression (including transcription and translation) of an IMQ nucleicacid sequence may be regulated with respect to the level of expression,the tissue type(s) where expression takes place and/or developmentalstage of expression. A number of heterologous regulatory sequences(e.g., promoters and enhancers) are available for controlling theexpression of an IMQ nucleic acid. These include constitutive, inducibleand regulatable promoters, as well as promoters and enhancers thatcontrol expression in a tissue- or temporal-specific manner. Exemplaryconstitutive promoters include the raspberry E4 promoter (U.S. Pat. Nos.5,783,393 and 5,783,394), the nopaline synthase (NOS) promoter (Ebert etal., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749, 1987), the octopinesynthase (OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol.Biol. 9:315-324, 1987) and the CaMV 35S promoter (Odell et al., Nature313:810-812, 1985 and Jones J D et al, 1992, Transgenic Res.,1:285-297), the figwort mosaic virus 35S-promoter (U.S. Pat. No.5,378,619), the light-inducible promoter from the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter(Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:6624-6628, 1987), thesucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. (U.S.A)87:4144-4148, 1990), the R gene complex promoter (Chandler et al., ThePlant Cell 1:1175-1183, 1989), the chlorophyll a/b binding protein genepromoter, the CsVMV promoter (Verdaguer B et al., 1998, Plant Mol Biol.,37:1055-1067), and the melon actin promoter (published PCT applicationWO0056863). Exemplary tissue-specific promoters include the tomato E4and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AII genepromoter (Van Haaren M J J et al., 1993, Plant Mol Bio., 21:625-640).

In one preferred embodiment, expression of the IMQ nucleic acid sequenceis under control of regulatory sequences from genes whose expression isassociated with early seed and/or embryo development. Indeed, in apreferred embodiment, the promoter used is a seed-enhanced promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219, 1991), globulin(Belanger and Kriz, Genet., 129: 863-872, 1991, GenBank Accession No.L22295), gamma zein Z 27 (Lopes et al., Mol Gen Genet., 247:603-613,1995), L3 oleosin promoter (U.S. Pat. No. 6,433,252), phaseolin (Bustoset al., Plant Cell, 1(9):839-853, 1989), arcelin5 (U.S. Application No.2003/0046727), a soybean 7S promoter, a 7Sα promoter (U.S. ApplicationNo. 2003/0093828), the soybean 7Sα′ beta conglycinin promoter, a 7S α′promoter (Beachy et al., EMBO J., 4:3047, 1985; Schuler et al., NucleicAcid Res., 10(24):8225-8244, 1982), soybean trypsin inhibitor (Riggs etal., Plant Cell 1(6):609-621, 1989), ACP (Baerson et al., Plant Mol.Biol., 22(2):255-267, 1993), stearoyl-ACP desaturase (Slocombe et al.,Plant Physiol. 104(4):167-176, 1994), soybean a′ subunit ofβ-conglycinin (Chen et al., Proc. Natl. Acad. Sci. 83:8560-8564, 1986),Vicia faba USP (P-Vf.Usp, SEQ ID NO: 1, 2, and 3 in (U.S. ApplicationNo. 2003/229918) and Zea mays L3 oleosin promoter (Hong et al., PlantMol. Biol., 34(3):549-555, 1997). Also included are the zeins, which area group of storage proteins found in corn endosperm. Genomic clones forzein genes have been isolated (Pedersen et al., Cell, 29:1015-1026,1982; and Russell et al., Transgenic Res. 6(2):157-168) and thepromoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD,27 kD and genes, could also be used. Other promoters known to function,for example, in corn include the promoters for the following genes:waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthases,debranching enzymes, oleosins, glutelins and sucrose synthases. Legumegenes whose promoters are associated with early seed and embryodevelopment include V. faba legumin (Baumlein et al., 1991, Mol. Gen.Genet. 225:121-8; Baumlein et al., 1992, Plant J. 2:233-9), V. faba usp(Fiedler et al., 1993, Plant Mol. Biol. 22:669-79), pea convicilin (Bownet al., 1988, Biochem. J. 251:717-26), pea lectin (dePater et al., 1993,Plant Cell 5:877-86), P. vulgaris beta phaseolin (Bustos et al., 1991,EMBO J. 10:1469-79), P. vulgaris DLEC2 and PHS [beta] (Bobb et al.,1997, Nucleic Acids Res. 25:641-7), and soybean beta-Conglycinin, 7Sstorage protein (Chamberland et al., 1992, Plant Mol. Biol. 19:937-49).

Cereal genes whose promoters are associated with early seed and embryodevelopment include rice glutelin (“GluA-3,” Yoshihara and Takaiwa,1996, Plant Cell Physiol. 37:107-11; “GluB-1,” Takaiwa et al., 1996,Plant Mol. Biol. 30:1207-21; Washida et al., 1999, Plant Mol. Biol.40:1-12; “Gt3,” Leisy et al., 1990, Plant Mol. Biol. 14:41-50), riceprolamin (Zhou & Fan, 1993, Transgenic Res. 2:141-6), wheat prolamin(Hammond-Kosack et al., 1993, EMBO 12:545-54), maize zein (Z4, Matzke etal., 1990, Plant Mol. Biol. 14:323-32), and barley B-hordeins (Entwistleet al., 1991, Plant Mol. Biol. 17:1217-31).

Other genes whose promoters are associated with early seed and embryodevelopment include oil palm GLO7A (7S globulin, Morcillo et al., 2001,Physiol. Plant 112:233-243), Brassica napus napin, 2S storage protein,and napA gene (Josefsson et al., 1987, 1 Biol. Chem. 262:12196-201;Stalberg et al., 1993, Plant Mol. Biol. 1993 23:671-83; Ellerstrom etal., 1996, Plant Mol. Biol. 32:1019-27), Brassica napus oleosin (Keddieet al., 1994, Plant Mol. Biol. 24:327-40), Arabidopsis oleosin (Plant etal., 1994, Plant Mol. Biol. 25:193-205), Arabidopsis FAE1 (Rossak etal., 2001, Plant Mol. Biol. 46:717-25), Canavalia gladiata conA(Yamamoto et al., 1995, Plant Mol. Biol. 27:729-41), and Catharanthusroseus strictosidine synthase (Str, Ouwerkerk and Memelink, 1999, Mol.Gen. Genet. 261:635-43). In another preferred embodiment, regulatorysequences from genes expressed during oil biosynthesis are used (see,e.g., U.S. Pat. No. 5,952,544). Alternative promoters are from plantstorage protein genes (Bevan et al., 1993, Philos. Trans. R. Soc. Lond.B. Biol. Sci. 342:209-15). Additional promoters that may be utilized aredescribed, for example, in U.S. Pat. Nos. 5,378,619; 5,391,725;5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441;5,633,435; and 4,633,436.

In yet another aspect, in some cases it may be desirable to inhibit theexpression of the endogenous IMQ nucleic acid sequence in a host cell.Exemplary methods for practicing this aspect of the disclosure include,but are not limited to antisense suppression (Smith, et al., 1988,Nature, 334:724-726; van der Krol et al., 1988, BioTechniques,6:958-976); co-suppression (Napoli, et al., 1990, Plant Cell,2:279-289); ribozymes (PCT Publication WO 97/10328); and combinations ofsense and antisense (Waterhouse, et al., 1998, Proc. Natl. Acad. Sci.USA, 95:13959-13964). Methods for the suppression of endogenoussequences in a host cell typically employ the transcription ortranscription and translation of at least a portion of the sequence tobe suppressed. Such sequences may be homologous to coding as well asnon-coding regions of the endogenous sequence. Antisense inhibition mayuse the entire cDNA sequence (Sheehy et al., 1988, Proc. Natl. Acad.Sci. USA, 85:8805-8809), a partial cDNA sequence including fragments of5′ coding sequence, (Cannon et al., 1990, Plant Mol. Biol., 15:39-47),or 3′ non-coding sequences (Ch'ng et al., 1989, Proc. Natl. Acad. Sci.USA, 86:10006-10010). Cosuppression techniques may use the entire cDNAsequence (Napoli et al., 1990, Plant Cell, 2:279-289; van der Krol etal., 1990, Plant Cell, 2:291-299), or a partial cDNA sequence (Smith etal., 1990, Mol. Gen. Genetics, 224:477-481).

Standard molecular and genetic tests may be performed to further analyzethe association between a nucleic acid sequence and an observedphenotype. Exemplary techniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include over-expression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing (VIGS; see, Baulcombe D, 1999,Arch. Virol. Suppl. 15:189-201).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science1995 270:467-470; Baldwin D et al., 1999, Cur. Opin. Plant Biol.2(2):96-103; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal N L etal., J Biotechnol. (2000) 78:271-280; Richmond T and Somerville S, Curr.Opin. Plant Biol. 2000 3:108-116). Expression profiling of individualtagged lines may be performed. Such analysis can identify other genesthat are coordinately regulated as a consequence of the over-expressionof the gene of interest, which may help to place an unknown gene in aparticular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with an Improved Oil Quantity Phenotypeand/or Improved Meal Quality Phenotype

Additional methods are disclosed herein of generating a plant having anIMQ and/or an IOQ phenotype, wherein a plant is identified that has anallele in its IMQ nucleic acid sequence that results in an IMQ phenotypeand/or an IOQ phenotype, compared to plants lacking the allele. Theplant can generate progeny, wherein the progeny inherit the allele andhave an IMQ phenotype and/or an IOQ phenotype. For example, providedherein is a method of identifying plants that have mutations in theendogenous IMQ nucleic acid sequence that confer an IMQ phenotype and/oran IOQ phenotype and generating progeny of these plants with an IMQand/or IOQ phenotype that are not genetically modified. In someembodiments, the plants have an IMQ phenotype with an altered proteinand/or fiber content or seed meal content, or an IOQ phenotype, with analtered oil content.

In one method, called “TILLING” (for targeting induced local lesions ingenomes), mutations are induced in the seed of a plant of interest, forexample, using EMS (ethylmethane sulfonate) treatment. The resultingplants are grown and self-fertilized, and the progeny are used toprepare DNA samples. PCR amplification and sequencing of the IMQ nucleicacid sequence is used to identify whether a mutated plant has a mutationin the IMQ nucleic acid sequence. Plants having IMQ mutations may thenbe tested for altered oil, protein, and/or fiber content, oralternatively, plants may be tested for altered oil, protein, and/orfiber content, and then PCR amplification and sequencing of the IMQnucleic acid sequence is used to determine whether a plant havingaltered oil, protein, and/or fiber content has a mutated IMQ nucleicacid sequence. TILLING can identify mutations that may alter theexpression of specific genes or the activity of proteins encoded bythese genes (see Colbert et al., 2001, Plant Physiol. 126:480-484;McCallum et al., 2000, Nature Biotechnology 18:455-457).

In another method, a candidate gene/Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in the IMQ nucleic acid sequence or orthologs ofthe IMQ nucleic acid sequence that may confer altered oil, protein,and/or fiber content (see Bert et al., Theor Appl Genet., 2003 June;107(1):181-9; and Lionneton et al., Genome, 2002 December;45(6):1203-15). Thus, in a further aspect of the disclosure, an IMQnucleic acid is used to identify whether a plant having altered oil,protein, and/or fiber content has a mutation an endogenous IMQ nucleicacid sequence or has a particular allele that causes altered oil,protein, and/or fiber content.

While the disclosure has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from thedisclosure. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with thedisclosure. All cited patents, patent applications, and sequenceinformation in referenced public databases are also incorporated byreference.

EXAMPLES Example 1 Generation of Plants with an IMQ Phenotype and/or anIOQ Phenotype by Transformation with an Activation Tagging Construct

This Example describes the generation of transgenic plants with alteredoil, protein, and/or fiber content.

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI#6537289; Weigel D et al., 2000, Plant Physiology,122:1003-1013). Standard methods were used for the generation ofArabidopsis transgenic plants, and were essentially as described inpublished application PCT WO0183697. Briefly, T0 Arabidopsis (Col-0)plants were transformed with Agrobacterium carrying the pSKI015 vector,which comprises T-DNA derived from the Agrobacterium Ti plasmid, anherbicide resistance selectable marker gene, and the 4×CaMV 35S enhancerelement. Transgenic plants were selected at the T1 generation based onherbicide resistance. T2 seed (from T1 plants) was harvested and sown insoil. T2 plants were exposed to the herbicide to kill plants lacking theACTTAG vector. T2 plants were grown to maturity, allowed toself-fertilize and set seed. T3 seed (from the T2 plants) was harvestedin bulk for each line.

T3 seed was analyzed by Near Infrared Spectroscopy (NIR) at the time ofharvest. NIR spectra were captured using a Bruker 22 near infraredspectrometer. Bruker Software was used to estimate total seed oil, totalseed protein and total seed fiber content using data from NIR analysisand reference methods according to the manufacturer's instructions. Oilcontent predicting calibrations were developed following the generalmethod of AOCS Procedure Aml-92, Official Methods and RecommendedPractices of the American Oil Chemists Society, 5th Ed., AOCS,Champaign, Ill. A NIR protein content predicting calibration wasdeveloped using total nitrogen content data of seed samples followingthe general method of Dumas Procedure AOAC 968.06 (Official Methods ofAnalysis of AOAC International 17^(th) Edition AOAC, Gaithersburg, Md.).A fiber content predicting calibration was developed by measuring crudefiber content in a set of seed samples. Fiber content of in a known massof seed was determined using the method of Honig and Rackis, (1979, JAgri. Food Chem., 27: 1262-1266). Digestible protein content of in aknown mass of seed was determined by quantifying the individual aminoacids liberated by an acid hydrolysis Steine and Moore (1958, Anal.Chem., 30:1185-1190). The quantification was performed by the AminoQuant (Agilent). The undigested protein remaining associated with thenon-digestible fraction is measured by the same method described for thewhole seed homogenate. Digestible protein content is determined bysubtracting the amount of undigested protein associated with thenon-digestible fraction from the total amount of protein in the seedsample.

Seed oil, protein, digestible protein and fiber values in 82,274 lineswere determined by NIR spectroscopy and normalized to allow comparisonof seed component values in plants grown at different times. Oil,protein and fiber values were normalized by calculating the average oil,protein and fiber values in seed from all plants planted on the same day(including a large number of other ACTTAG plants, including control,wild-type, or non-transgenic plants). The seed components for each linewas expressed as a “percent relative value” which was calculated bydividing the component value for each line with the average componentvalue for all lines planted on the same day (which should approximatethe value in control, wild-type, or non-transgenic plants). The “percentrelative protein” and “percent relative fiber” were calculatedsimilarly.

Inverse PCR was used to recover genomic DNA flanking the T-DNAinsertion. The PCR product was subjected to sequence analysis and placedon the genome using a basic BLASTN search and/or a search of theArabidopsis Information Resource (TAIR) database (available at thepublicly available website). Promoters within 9 kb of the enhancers inthe ACTTAG element are considered to be within “activation space.” Geneswith T-DNA inserts within coding sequences were not considered to bewithin “activation space.” The ACTTAG lines with the above average oiland protein values, and below average fiber values were identified andare listed in column 3 of Table 1.

TABLE 1 4. Relative 5. Relative 6. Relative 1. Gene 3. ACTTAG SeedProtein Seed Fiber Seed Oil 7. alias 2. Tair Line Content ContentContent GC FA IMQ34.3 At2g31480 W000137133 135.45% 89.55% 82.65% IMQ34.4At2g31490 W000137133 135.45% 89.55% 82.65% IMQ35.1 At2g34030 W000041983122.86% 92.93% 80.44% IMQ35.2 At2g34040 W000041983 122.86% 92.93% 80.44%IMQ35.2 At2g34040 W000041983 122.86% 92.93% 80.44% IMQ35.3 At2g34050W000041983 122.86% 92.93% 80.44% IMQ35.4 At2g34060 W000041983 122.86%92.93% 80.44% IMQ36.1 At2g34400 W000146178 109.53% 93.15% 94.98% IMQ36.2At2g34410 W000146178 109.53% 93.15% 94.98% IMQ36.2 At2g34410 W000146178109.53% 93.15% 94.98% IMQ36.2 At2g34410 W000146178 109.53% 93.15% 94.98%IMQ36.3 At2g34420 W000146178 109.53% 93.15% 94.98% IMQ36.3 At2g34420W000146178 109.53% 93.15% 94.98% IMQ36.4 At2g34430 W000146178 109.53%93.15% 94.98% IMQ36.5 At2g34440 W000146178 109.53% 93.15% 94.98% IMQ36.6At2g34450 W000146178 109.53% 93.15% 94.98% IMQ36.6 At2g34450 W000146178109.53% 93.15% 94.98% IMQ37.1 At3g03800 W000168536 105.07% 88.55% 98.42%IMQ37.2 At3g03810 W000168536 105.07% 88.55% 98.42% IMQ37.3 At3g03820W000168536 105.07% 88.55% 98.42% IMQ37.4 At3g03830 W000168536 105.07%88.55% 98.42% IMQ37.5 At3g03840 W000168536 105.07% 88.55% 98.42% IMQ37.6At3g03847 W000168536 105.07% 88.55% 98.42% 97.87% IMQ37.7 At3g03850W000168536 105.07% 88.55% 98.42% IMQ38.1 At3g07100 W000182714 101.56%90.13% 99.68% IMQ38.2 At3g07110 W000182714 101.56% 90.13% 99.68% IMQ38.2At3g07110 W000182714 101.56% 90.13% 99.68% IMQ38.3 At3g07120 W000182714101.56% 90.13% 99.68% IMQ38.4 At3g07130 W000182714 101.56% 90.13% 99.68%IMQ38.5 At3g07140 W000182714 101.56% 90.13% 99.68% IMQ38.5 At3g07140W000182714 101.56% 90.13% 99.68% IMQ38.6 At3g07150 W000182714 101.56%90.13% 99.68% IMQ39.1 At3g15480 W000190425 151.85% 106.23% 57.69%IMQ39.2 At3g15490 W000190425 151.85% 106.23% 57.69% 69.66% IMQ39.3At3g15500 W000190425 151.85% 106.23% 57.69% IMQ40.1 At3g16580 W000085366119.68% 89.13% 84.56% 99.63% IMQ40.2 At3g16590 W000085366 119.68% 89.13%84.56% IMQ40.3 At3g16600 W000085366 119.68% 89.13% 84.56% IMQ40.4At3g16610 W000085366 119.68% 89.13% 84.56% IMQ40.5 At3g16620 W000085366119.68% 89.13% 84.56% IMQ41.1 At3g17640 W000192636 101.54% 86.92%102.79% 107.27% IMQ41.2 At3g17650 W000192636 101.54% 86.92% 102.79%IMQ41.3 At3g17660 W000192636 101.54% 86.92% 102.79% IMQ41.4 At3g17670W000192636 101.54% 86.92% 102.79% IMQ41.5 At3g17680 W000192636 101.54%86.92% 102.79% 107.27% IMQ41.5 At3g17680 W000192636 101.54% 86.92%102.79% IMQ41.6 At3g17690 W000192636 101.54% 86.92% 102.79% 107.27%IMQ42.1 At3g19850 W000093675 112.46% 94.21% 95.25% IMQ42.2 At3g19860W000093675 112.46% 94.21% 95.25% IMQ42.2 At3g19860 W000093675 112.46%94.21% 95.25%

TABLE 2 7. Putative 3. Nucleic biochemical 1. Gene Acid seq. 4. SEQ 5.Polypeptide 6. SEQ function/protein 8. Conserved alias 2. Tair GI# ID NOseq. GI# ID NO name protein domain IMQ34.3 At2g31480 gi|42569515 SEQ IDgi|42569516 SEQ ID unknown protein NO: 1 NO: 2 IMQ34.4 At2g31490gi|18402681 SEQ ID gi|18402682 SEQ ID unknown protein NO: 3 NO: 4IMQ35.1 At2g34030 gi|42569600 SEQ ID gi|42569601 SEQ ID calcium ionbinding IPR002048 Calcium- NO: 5 NO: 6 binding EF-hand IMQ35.2 At2g34040gi|30685905 SEQ ID gi|30685906 SEQ ID unknown protein IPR008383Apoptosis NO: 7 NO: 8 inhibitory 5 IMQ35.2 At2g34040 gi|30685899 SEQ IDgi|18403429 SEQ ID unknown protein IPR008383 Apoptosis NO: 9 NO: 10inhibitory 5 IMQ35.3 At2g34050 gi|30685908 SEQ ID gi|18403431 SEQ IDunknown protein IPR010591 ATP11 NO: 11 NO: 12 IMQ35.4 At2g34060gi|30685912 SEQ ID gi|15226205 SEQ ID peroxidase IPR002016 Haem NO: 13NO: 14 peroxidase, plant/fungal/bacterial; IPR000823 Plant peroxidaseIMQ36.1 At2g34400 gi|18403542 SEQ ID gi|15226301 SEQ ID unknown proteinIPR002885 NO: 15 NO: 16 Pentatricopeptide repeat; IPR008940 Proteinprenyltransferase IMQ36.2 At2g34410 gi|79324292 SEQ ID gi|79324293 SEQID O-acetyltransferase IPR012419 Cas1p-like NO: 17 NO: 18 IMQ36.2At2g34410 gi|79324284 SEQ ID gi|79324285 SEQ ID O-acetyltransferaseIPR012419 Cas1p-like NO: 19 NO: 20 IMQ36.2 At2g34410 gi|42569608 SEQ IDgi|42569609 SEQ ID O-acetyltransferase IPR012419 Cas1p-like NO: 21 NO:22 IMQ36.3 At2g34420 gi|30686050 SEQ ID gi|30686051 SEQ ID LHB1B2IPR001344 Chlorophyll NO: 23 NO: 24 A-B binding protein IMQ36.3At2g34420 gi|30686047 SEQ ID gi|18403546 SEQ ID LHB1B2 IPR001344Chlorophyll NO: 25 NO: 26 A-B binding protein IMQ36.4 At2g34430gi|30686053 SEQ ID gi|18403549 SEQ ID LHB1B1; chlorophyll IPR001344Chlorophyll NO: 27 NO: 28 binding A-B binding protein IMQ36.5 At2g34440gi|18403551 SEQ ID gi|15226309 SEQ ID transcription factor IPR002100Transcription NO: 29 NO: 30 factor, MADS-box IMQ36.6 At2g34450gi|79324302 SEQ ID gi|79324303 SEQ ID unknown protein IPR000910 HMG1/2NO: 31 NO: 32 (high mobility group) box IMQ36.6 At2g34450 gi|30686062SEQ ID gi|18403553 SEQ ID transcription factor IPR000910 HMG1/2 NO: 33NO: 34 (high mobility group) box IMQ37.1 At3g03800 gi|18396673 SEQ IDgi|15228637 SEQ ID SYP131; t-SNARE IPR000727 Target NO: 35 NO: 36 SNAREcoiled-coil region; IPR006011 Syntaxin, N-terminal; IPR006012Syntaxin/epimorphin family IMQ37.2 At3g03810 gi|30678945 SEQ IDgi|30678946 SEQ ID unknown protein IPR004348 Hypothetical NO: 37 NO: 38plant protein IMQ37.3 At3g03820 gi|18396679 SEQ ID gi|15228639 SEQ IDunknown protein IPR003676 Auxin NO: 39 NO: 40 responsive SAUR proteinIMQ37.4 At3g03830 gi|30678954 SEQ ID gi|15228640 SEQ ID unknown proteinIPR003676 Auxin NO: 41 NO: 42 responsive SAUR protein IMQ37.5 At3g03840gi|42563470 SEQ ID gi|15228641 SEQ ID unknown protein IPR003676 AuxinNO: 43 NO: 44 responsive SAUR protein IMQ37.6 At3g03847 gi|22330828 SEQID gi|22330829 SEQ ID unknown protein IPR003676 Auxin NO: 45 NO: 46responsive SAUR protein IMQ37.7 At3g03850 gi|30678958 SEQ ID gi|30678959SEQ ID unknown protein NO: 47 NO: 48 IMQ38.1 At3g07100 gi|30680128 SEQID gi|30680129 SEQ ID protein binding/ IPR006896 NO: 49 NO: 50transporter Sec23/Sec24 trunk region; IPR006900 Sec23/Sec24 helicalregion; IPR006895 Zinc finger, Sec23/Sec24-type; IPR006706 Extensin-likeregion; IPR007123 Gelsolin region IMQ38.2 At3g07110 gi|79313148 SEQ IDgi|79313149 SEQ ID structural IPR005822 Ribosomal NO: 51 NO: 52constituent protein L13; of ribosome IPR005755 Ribosomal protein L13,archea and eukaryotic form; IPR005823 Ribosomal protein L13, bacterialand organelle form IMQ38.2 At3g07110 gi|30680131 SEQ ID gi|15231394 SEQID structural IPR005822 Ribosomal NO: 53 NO: 54 constituent protein L13;of ribosome IPR005755 Ribosomal protein L13, archea and eukaryotic form;IPR005823 Ribosomal protein L13, bacterial and organelle form IMQ38.3At3g07120 gi|30680134 SEQ ID gi|15231396 SEQ ID protein binding/IPR001841 Zinc finger, NO: 55 NO: 56 ubiquitin-protein RING-typeligase/zinc ion binding IMQ38.4 At3g07130 gi|42563571 SEQ ID gi|15231398SEQ ID hydrolase/protein IPR004843 NO: 57 NO: 58 serine/threonineMetallophosphoesterase phosphatase IMQ38.5 At3g07140 gi|42570456 SEQ IDgi|30680143 SEQ ID GPI-anchor IPR007245 Gpi16 NO: 59 NO: 60 transamidasesubunit, GPI transamidase component IMQ38.5 At3g07140 gi|30680141 SEQ IDgi|18397852 SEQ ID GPI-anchor IPR007245 Gpi16 NO: 61 NO: 62 transamidasesubunit, GPI transamidase component IMQ38.6 At3g07150 gi|18397855 SEQ IDgi|15231401 SEQ ID unknown protein NO: 63 NO: 64 IMQ39.1 At3g15480gi|30683621 SEQ ID gi|18400781 SEQ ID unknown protein IPR009606 Proteinof NO: 65 NO: 66 unknown function DUF1218 IMQ39.2 At3g15490 gi|42564187SEQ ID gi|42564188 SEQ ID unknown protein IPR005061 Protein of NO: 67NO: 68 unknown function DUF292, eukaryotic IMQ39.3 At3g15500 gi|30683631SEQ ID gi|15232604 SEQ ID ATNAC3; IPR003441 No apical NO: 69 NO: 70transcription meristem (NAM) protein factor IMQ40.1 At3g16580gi|30684147 SEQ ID gi|18401179 SEQ ID unknown protein IPR001810Cyclin-like NO: 71 NO: 72 F-box; IPR006527 F-box protein interactiondomain IMQ40.2 At3g16590 gi|18401181 SEQ ID gi|15228255 SEQ ID unknownprotein IPR006527 F-box protein NO: 73 NO: 74 interaction domain;IPR001810 Cyclin-like F-box IMQ40.3 At3g16600 gi|18401188 SEQ IDgi|15228256 SEQ ID ATP binding/ATP- IPR000330 SNF2- NO: 75 NO: 76dependent helicase/ related; DNA binding/ IPR001410 DEAD/DEAHhelicase/nucleic box helicase; acid binding/protein IPR001650 Helicase,binding/ubiquitin- C-terminal protein ligase/zinc ion binding IMQ40.4At3g16610 gi|18401191 SEQ ID gi|15228257 SEQ ID unknown proteinIPR001841 Zinc finger, NO: 77 NO: 78 RING-type; IPR002885Pentatricopeptide repeat IMQ40.5 At3g16620 gi|30684161 SEQ IDgi|15228272 SEQ ID ATTOC120; IPR006703 AIG1; NO: 79 NO: 80 proteinIPR002917 GTP-binding translocase protein, HSR1-related; IPR005688Chloroplast protein import component Toc34; IPR005690 Chloroplastprotein import component Toc86/159 IMQ41.1 At3g17640 gi|18401587 SEQ IDgi|15229088 SEQ ID protein binding IPR001611 Leucine-rich NO: 81 NO: 82repeat; IPR007090 Leucine-rich repeat, plant specific IMQ41.2 At3g17650gi|18401589 SEQ ID gi|18401590 SEQ ID PDE321; IPR004813 Oligopeptide NO:83 NO: 84 oligopeptide transporter OPT transporter superfamily;IPR004814 Oligopeptide transporter OPT; IPR004648 Tetrapeptidetransporter, OPT1/isp4 IMQ41.3 At3g17660 gi|18401594 SEQ ID gi|15229090SEQ ID DNA binding IPR001164 Arf GTPase NO: 85 NO: 86 activating proteinIMQ41.4 At3g17670 gi|18401598 SEQ ID gi|15229091 SEQ ID unknown proteinIPR001440 TPR repeat NO: 87 NO: 88 IMQ41.5 At3g17680 gi|79313276 SEQ IDgi|79313277 SEQ ID unknown protein IPR011684 KIP1-like NO: 89 NO: 90IMQ41.5 At3g17680 gi|30684630 SEQ ID gi|30684631 SEQ ID unknown proteinIPR011684 KIP1-like NO: 91 NO: 92 IMQ41.6 At3g17690 gi|30684635 SEQ IDgi|15229093 SEQ ID ATCNGC19; IPR005821 Ion transport NO: 93 NO: 94calmodulin binding/ protein; cyclic nucleotide IPR000595 Cyclicbinding/ion channel nucleotide-binding IMQ42.1 At3g19850 gi|42565033 SEQID gi|42565034 SEQ ID protein binding/ IPR004249 NPH3; NO: 95 NO: 96signal transducer IPR000210 BTB/POZ IMQ42.2 At3g19860 gi|79313298 SEQ IDgi|79313299 SEQ ID DNA binding IPR001092 Basic helix- NO: 97 NO: 98loop-helix dimerisation region bHLH; IPR003106 Leucine zipper, homeobox-associated IMQ42.2 At3g19860 gi|30685522 SEQ ID gi|15230975 SEQ ID DNAbinding/ IPR001092 Basic helix- NO: 99 NO: 100 transcription factorloop-helix dimerisation region bHLH; IPR003106 Leucine zipper, homeobox-associated

TABLE 3 5. Orthologous Genes: Nucleic 3. Nucleic Acid/Polypeptide seq.GI# 1. Gene Acid seq. 4. Polypeptide Nucleic Acid Polypeptide alias 2.Tair GI# seq. GI# GI# GI# Species IMQ34.3 At2g31480 gi|42569515gi|42569516 IMQ34.4 At2g31490 gi|18402681 gi|18402682 gi|15146360gi|57899156 Oryza sativa (japonica cultivar-group) gi|34910867gi|34910868 Oryza sativa (japonica cultivar-group) gi|34909561gi|34909562 Oryza sativa (japonica cultivar-group) IMQ35.1 At2g34030gi|42569600 gi|42569601 gi|30690570 gi|15218787 Arabidopsis thalianagi|18403422 gi|15226191 Arabidopsis thaliana gi|30695447 gi|30695448Arabidopsis thaliana IMQ35.2 At2g34040 gi|30685905 gi|30685906gi|30685899 gi|18403429 Arabidopsis thaliana gi|42562386 gi|42562387Arabidopsis thaliana gi|50907460 gi|50907461 Oryza sativa (japonicacultivar-group) gi|66526466 gi|66526467 Apis mellifera IMQ35.2 At2g34040gi|30685899 gi|18403429 gi|30685905 gi|30685906 Arabidopsis thalianagi|42562386 gi|42562387 Arabidopsis thaliana gi|50907460 gi|50907461Oryza sativa (japonica cultivar-group) gi|66526466 gi|66526467 Apismellifera IMQ35.3 At2g34050 gi|30685908 gi|18403431 gi|50907446gi|50907447 Oryza sativa (japonica cultivar-group) gi|66814293gi|66814294 Dictyostelium discoideum gi|50751555 gi|50751556 Gallusgallus IMQ35.4 At2g34060 gi|30685912 gi|15226205 gi|50918940 gi|50918941Oryza sativa (japonica cultivar-group) gi|55701112 gi|55701113 Oryzasativa (japonica cultivar-group) gi|537318 gi|537319 Medicago sativaIMQ36.1 At2g34400 gi|18403542 gi|15226301 gi|18398256 gi|15231970Arabidopsis thaliana gi|18399739 gi|15230593 Arabidopsis thalianagi|18420159 gi|15233645 Arabidopsis thaliana IMQ36.2 At2g34410gi|79324292 gi|79324293 gi|79324284 gi|79324285 Arabidopsis thalianagi|42569608 gi|42569609 Arabidopsis thaliana gi|30694937 gi|18422663Arabidopsis thaliana gi|42562401 gi|42562402 Arabidopsis thalianagi|20161482 gi|55297065 Oryza sativa (japonica cultivar-group) IMQ36.2At2g34410 gi|79324284 gi|79324285 gi|79324292 gi|79324293 Arabidopsisthaliana gi|42569608 gi|42569609 Arabidopsis thaliana gi|30694937gi|18422663 Arabidopsis thaliana gi|42562401 gi|42562402 Arabidopsisthaliana gi|20161482 gi|55297065 Oryza sativa (japonica cultivar-group)IMQ36.2 At2g34410 gi|42569608 gi|42569609 gi|79324292 gi|79324293Arabidopsis thaliana gi|79324284 gi|79324285 Arabidopsis thalianagi|30694937 gi|18422663 Arabidopsis thaliana gi|42562401 gi|42562402Arabidopsis thaliana gi|20161482 gi|55297065 Oryza sativa (japonicacultivar-group) IMQ36.3 At2g34420 gi|30686050 gi|30686051 gi|30686047gi|18403546 Arabidopsis thaliana gi|30690951 gi|15220615 Arabidopsisthaliana gi|30690948 gi|18397288 Arabidopsis thaliana gi|30690947gi|18397286 Arabidopsis thaliana IMQ36.3 At2g34420 gi|30686047gi|18403546 gi|30690951 gi|15220615 Arabidopsis thaliana gi|30690948gi|18397288 Arabidopsis thaliana gi|30690947 gi|18397286 Arabidopsisthaliana gi|31323255 gi|31323256 Brassica oleracea IMQ36.4 At2g34430gi|30686053 gi|18403549 gi|21137 gi|21138 Sinapis alba gi|18266038gi|18266039 Brassica oleracea gi|30690951 gi|15220615 Arabidopsisthaliana IMQ36.5 At2g34440 gi|18403551 gi|15226309 gi|18397662gi|15230767 Arabidopsis thaliana gi|30682570 gi|30682571 Arabidopsisthaliana gi|18424355 gi|15239333 Arabidopsis thaliana IMQ36.6 At2g34450gi|79324302 gi|79324303 gi|30686062 gi|18403553 Arabidopsis thalianagi|20161570 gi|56202161 Oryza sativa (japonica cultivar-group)gi|63020535 gi|63020536 Cucumis sativus gi|34910913 gi|34910914 Oryzasativa (japonica cultivar-group) IMQ36.6 At2g34450 gi|30686062gi|18403553 gi|20161570 gi|56202161 Oryza sativa (japonicacultivar-group) gi|63020535 gi|63020536 Cucumis salivus gi|42562198gi|18394900 Arabidopsis thaliana IMQ37.1 At3g03800 gi|18396673gi|15228637 gi|18415700 gi|18415701 Arabidopsis thaliana gi|51963279gi|51963280 Oryza sativa (japonica cultivar-group) gi|50934370gi|50934371 Oryza sativa (japonica cultivar-group) gi|55741415gi|55741416 Oryza sativa (japonica cultivar-group) IMQ37.2 At3g03810gi|30678945 gi|30678946 gi|46063406 gi|50878393 Oryza sativa (japonicacultivar-group) gi|30689847 gi|30689848 Arabidopsis thaliana gi|30679041gi|22329335 Arabidopsis thaliana IMQ37.3 At3g03820 gi|18396679gi|15228639 gi|42563470 gi|15228641 Arabidopsis thaliana gi|30678958gi|30678959 Arabidopsis thaliana gi|30678954 gi|15228640 Arabidopsisthaliana IMQ37.4 At3g03830 gi|30678954 gi|15228640 gi|30678958gi|30678959 Arabidopsis thaliana gi|18396679 gi|15228639 Arabidopsisthaliana gi|42563470 gi|15228641 Arabidopsis thaliana IMQ37.5 At3g03840gi|42563470 gi|15228641 gi|18396679 gi|15228639 Arabidopsis thalianagi|30678958 gi|30678959 Arabidopsis thaliana gi|30678954 gi|15228640Arabidopsis thaliana IMQ37.6 At3g03847 gi|22330828 gi|22330829gi|42567926 gi|15238721 Arabidopsis thaliana gi|42567924 gi|15238716Arabidopsis thaliana gi|30686621 gi|15238715 Arabidopsis thalianaIMQ37.7 At3g03850 gi|30678958 gi|30678959 gi|30678954 gi|15228640Arabidopsis thaliana gi|42563470 gi|15228641 Arabidopsis thalianagi|18396679 gi|15228639 Arabidopsis thaliana IMQ38.1 At3g07100gi|30680128 gi|30680129 gi|50921574 gi|50921575 Oryza sativa (japonicacultivar-group) gi|74199102 gi|74199103 Mus musculus gi|31340743gi|30424898 Mus musculus IMQ38.2 At3g07110 gi|79313148 gi|79313149gi|30680131 gi|15231394 Arabidopsis thaliana gi|30695535 gi|15239698Arabidopsis thaliana gi|30687674 gi|15230197 Arabidopsis thalianagi|42566745 gi|15235617 Arabidopsis thaliana IMQ38.2 At3g07110gi|30680131 gi|15231394 gi|30695535 gi|15239698 Arabidopsis thalianagi|30687674 gi|15230197 Arabidopsis thaliana gi|42566745 gi|15235617Arabidopsis thaliana IMQ38.3 At3g07120 gi|30680134 gi|15231396gi|42565172 gi|15230243 Arabidopsis thaliana gi|42570141 gi|42570142Arabidopsis thaliana gi|18413925 gi|15235589 Arabidopsis thalianaIMQ38.4 At3g07130 gi|42563571 gi|15231398 gi|13925770 gi|13925771Glycine max gi|62177682 gi|62177683 Medicago truncatula gi|50920094gi|50920095 Oryza sativa (japonica cultivar-group) IMQ38.5 At3g07140gi|42570456 gi|30680143 gi|30680141 gi|18397852 Arabidopsis thalianagi|77548247 gi|77550830 Oryza sativa (japonica cultivar-group)gi|73992512 gi|73992513 Canis familiaris IMQ38.5 At3g07140 gi|30680141gi|18397852 gi|42570456 gi|30680143 Arabidopsis thaliana gi|77548247gi|77550830 Oryza sativa (japonica cultivar-group) gi|73992512gi|73992513 Canis familiaris IMQ38.6 At3g07150 gi|18397855 gi|15231401gi|19386744 gi|57900451 Oryza sativa (japonica cultivar-group)gi|34907011 gi|34907012 Oryza sativa (japonica cultivar-group) IMQ39.1At3g15480 gi|30683621 gi|18400781 gi|42562716 gi|18404242 Arabidopsisthaliana gi|38016520 gi|38016521 Gossypium barbadense gi|50936920gi|50936921 Oryza sativa (japonica cultivar-group) IMQ39.2 At3g15490gi|42564187 gi|42564188 gi|18397647 gi|15226014 Arabidopsis thalianagi|50918206 gi|50918207 Oryza sativa (japonica cultivar-group)gi|30683233 gi|15222251 Arabidopsis thaliana IMQ39.3 At3g15500gi|30683631 gi|15232604 gi|42562715 gi|15219112 Arabidopsis thalianagi|66394515 gi|66394516 Glycine max gi|6175245 gi|6175246 Lycopersiconesculentum IMQ40.1 At3g16580 gi|30684147 gi|18401179 gi|42569040gi|15225992 Arabidopsis thaliana gi|18398432 gi|15223198 Arabidopsisthaliana gi|18401705 gi|18401706 Arabidopsis thaliana IMQ40.2 At3g16590gi|18401181 gi|15228255 gi|18391359 gi|15221240 Arabidopsis thalianagi|30684644 gi|18401609 Arabidopsis thaliana gi|18391361 gi|15221246Arabidopsis thaliana IMQ40.3 At3g16600 gi|18401188 gi|15228256gi|18403060 gi|18403061 Arabidopsis thaliana gi|18402451 gi|15231009Arabidopsis thaliana gi|51964729 gi|51964730 Oryza sativa (japonicacultivar-group) IMQ40.4 At3g16610 gi|18401191 gi|15228257 gi|50941690gi|50941691 Oryza sativa (japonica cultivar-group) gi|18483227gi|18483237 Sorghum bicolor gi|34906109 gi|34906110 Oryza sativa(japonica cultivar-group) gi|28564706 gi|57899529 Oryza sativa (japonicacultivar-group) IMQ40.5 At3g16620 gi|30684161 gi|15228272 gi|30679717gi|15227268 Arabidopsis thaliana gi|37536637 gi|37536638 Oryza sativa(japonica cultivar-group) gi|44662984 gi|44662985 Physcomitrella patensIMQ41.1 At3g17640 gi|18401587 gi|15229088 gi|50931366 gi|50931367 Oryzasativa (japonica cultivar-group) gi|51339056 gi|55733924 Oryza sativa(japonica cultivar-group) gi|30678565 gi|30678566 Arabidopsis thalianaIMQ41.2 At3g17650 gi|18401589 gi|18401590 gi|30694308 gi|18402162Arabidopsis thaliana gi|42562981 gi|15218799 Arabidopsis thalianagi|74267415 gi|74267416 Oryza sativa (japonica cultivar-group) IMQ41.3At3g17660 gi|18401594 gi|15229090 gi|30696440 gi|18423615 Arabidopsisthaliana gi|50510177 gi|50510178 Oryza sativa (japonica cultivar-group)gi|60596222 gi|76152990 Schistosoma japonicum IMQ41.4 At3g17670gi|18401598 gi|15229091 gi|53749471 gi|53749491 Solanum demissumgi|50929586 gi|50929587 Oryza sativa (japonica cultivar-group)gi|50928358 gi|50928359 Oryza sativa (japonica cultivar-group) IMQ41.5At3g17680 gi|79313276 gi|79313277 gi|30684630 gi|30684631 Arabidopsisthaliana gi|22330106 gi|22330107 Arabidopsis thaliana gi|53749471gi|53749473 Solanum demissum gi|50916296 gi|50916297 Oryza sativa(japonica cultivar-group) IMQ41.5 At3g17680 gi|30684630 gi|30684631gi|22330106 gi|22330107 Arabidopsis thaliana gi|53749471 gi|53749473Solanum demissum gi|50916296 gi|50916297 Oryza sativa (japonicacultivar-group) IMQ41.6 At3g17690 gi|30684635 gi|15229093 gi|42564623gi|18401606 Arabidopsis thaliana gi|50913256 gi|50913257 Oryza sativa(japonica cultivar-group) gi|51091189 gi|51091194 Oryza sativa (japonicacultivar-group) IMQ42.1 At3g19850 gi|42565033 gi|42565034 gi|22330149gi|22330150 Arabidopsis thaliana gi|50929590 gi|50929591 Oryza sativa(japonica cultivar-group) gi|50938220 gi|50938221 Oryza sativa (japonicacultivar-group) IMQ42.2 At3g19860 gi|79313298 gi|79313299 gi|30685522gi|15230975 Arabidopsis thaliana gi|51536177 gi|51536178 Oryza sativa(japonica cultivar-group) gi|30690567 gi|30690568 Arabidopsis thalianagi|22329203 gi|22329204 Arabidopsis thaliana IMQ42.2 At3g19860gi|30685522 gi|15230975 gi|51536177 gi|51536178 Oryza sativa (japonicacultivar-group) gi|22329203 gi|22329204 Arabidopsis thaliana gi|30690567gi|30690568 Arabidopsis thaliana

Example 2 Analysis of the Arabidopsis IMQ Sequence

Sequence analyses were performed with BLAST (Altschul et al., 1990, J.Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999, Nucleic Acids Res.27:260-262), INTERPRO (Mulder et al. 2003 Nucleic Acids Res. 31,315-318.), PSORT (Nakai K, and Horton P, 1999, Trends Biochem. Sci.24:34-6), and/or CLUSTAL (Thompson J D et al., 1994, Nucleic Acids Res.22:4673-4680). Conserved domains for each protein are listed in column 8of Table 2.

Example 3

To test whether over-expression of the genes in Tables 1 and 2 alter theseed composition phenotype, protein, digestible protein, oil and fibercontent in seeds from transgenic plants expressing these genes wascompared with protein, digestible protein, oil and fiber content inseeds from non-transgenic control plants. To do this, the genes werecloned into plant transformation vectors behind the strong constitutiveCsVMV promoter and the seed specific PRU promoter. These constructs weretransformed into Arabidopsis plants using the floral dip method. Theplant transformation vector contains a gene, which provides resistanceto a toxic compound, and serves as a selectable marker. Seed from thetransformed plants were plated on agar medium containing the toxiccompound. After 7 days, transgenic plants were identified as healthygreen plants and transplanted to soil. Non-transgenic control plantswere germinated on agar medium, allowed to grow for 7 days and thentransplanted to soil. Transgenic seedlings and non-transgenic controlplants were transplanted to two inch pots that were placed in randompositions in a 10 inch by 20 inch tray. The plants were grown tomaturity, allowed to self-fertilize and set seed. Seed was harvestedfrom each plant and its oil content estimated by Near Infrared (NIR)Spectroscopy using methods previously described. The effect of eachconstruct on seed composition was examined in at least two experiments.

Table 4 lists constructs tested for causing a significant increase inoil, protein, digestible protein or a significant decrease in fiber wereidentified by a two-way Analysis of Variance (ANOVA) test at a p-value≦0.05. The ANOVA p-values for Protein, Oil, Digestible Protein and Fiberare listed in columns 4-7, respectively. Those with a significantp-value are listed in bold. The Average values for Protein, Oil,Digestible Protein and Fiber are listed in columns 8-11, respectivelyand were calculated by averaging the average values determined for thetransgenic plants in each experiment.

TABLE 4 4. 5. 6. ANOVA 7. 10. ANOVA ANOVA Digestible ANOVA Digestible11. 1. Gene 2. TAIR 3. Construct Protein Oil Protein Fiber 8. Protein 9.Oil Protein Fiber IMQ37.5 At3g03840 CsVMV::At3g03840 0.433 0.415 0.0330.197 101.7% 98.7% 102.4% 98.7% IMQ37.6 At3g03847 CsVMV::At3g03847 0.9980.503 0.634 0.864 100.2% 99.4% 100.5% 99.8% IMQ37.6 At3g03847Pru::At3g03847 0.007 0.002 0.115 0.547 104.6% 94.4% 101.2% 100.6%IMQ39.1 At3g15480 Pru::At3g15480 0.521 0.450 0.026 0.011 100.9% 100.4%101.4% 97.6% IMQ39.2 At3g15490 CsVMV::At3g15490 0.192 0.463 0.176 0.035103.1% 99.1% 101.7% 97.8% IMQ39.2 At3g15490 Pru::At3g15490 0.125 0.551<0.0001 <0.0001 100.9% 99.4% 101.4% 97.8% IMQ40.1 At3g16580CsVMV::At3g16580 0.436 0.321 0.050 0.173 101.3% 99.0% 101.6% 98.9%IMQ40.1 At3g16580 Pru::At3g16580 0.269 0.972 0.005 0.003 101.3% 99.7%101.7% 97.4% IMQ40.3 At3g16600 Pru::At3g16600 0.019 0.019 0.220 0.136102.8% 97.0% 99.5% 101.5% IMQ41.1 At3g17640 CsVMV::At3g17640 0.991 0.6710.864 0.725 99.9% 99.5% 99.8% 99.8% IMQ41.1 At3g17640 Pru::At3g176400.024 0.006 0.041 0.003 97.1% 105.0% 101.3% 96.8% IMQ41.5 At3g17680CsVMV::At3g17680 0.567 0.873 0.803 0.290 101.3% 100.2% 100.3% 98.9%IMQ41.5 At3g17680 Pru::At3g17680 0.569 0.252 0.009 0.004 99.3% 101.8%102.1% 96.7% IMQ41.6 At3g17690 CsVMV::At3g17690 0.388 0.856 0.060 0.039102.7% 99.8% 102.4% 97.3% IMQ41.6 At3g17690 Pru::At3g17690 0.263 0.7260.003 0.009 102.0% 99.2% 103.0% 96.5%

Example 4

To test whether over-expression of the genes identified in Tables 1-4alter the seed composition phenotype, protein, digestible protein, oil,and fiber content in seeds from transgenic plants expressing these genesis compared with protein, digestible protein, oil and fiber content inseeds from non-transgenic control plants. Any one of the genesidentified in Tables 1-4 is used to transform Brassica napus (canola).To do this, the genes are cloned into plant transformation vectorsbehind the strong constitutive CsVMV promoter and the seed specificphaseolin promoter. These constructs (which include a gene encoding aselection agent) are transformed into canola plants.

Transformation of canola is accomplished via Agrobacterium-mediatedtransformation. Seeds are surface-sterilized with 10% commercial bleachfor 10 minutes and rinsed 3 times with sterile distilled water. Theseeds are then placed on one half concentration of MS basal medium(Murashige and Skoog, Physiol. Plant. 15:473-497, 1962) and maintainedunder growth regime set at 25° C., and a photoperiod of 16 hrs light/8hrs dark.

Hypocotyl segments (3-5 mm) are excised from 5-7 day old seedlings andplaced on callus induction medium K1D1 (MS medium with 1 mg/l kinetinand 1 mg/l 2,4-D) for 3 days as pre-treatment. The segments are thentransferred into a petri plate, treated with Agrobacterium Z7075 orLBA4404 strain containing pDAB721. The Agrobacterium is grown overnightat 28° C. in the dark on a shaker at 150 rpm and subsequentlyre-suspended in the culture medium.

After 30 minute treatment of the hypocotyl segments with Agrobacterium,these are placed back on the callus induction medium for 3 days.Following co-cultivation, the segments are placed on K1D1TC (callusinduction medium containing 250 mg/l Carbenicillin and 300 mg/lTimentin) for one week of recovery. Alternately, the segments are placeddirectly on selection medium K1D1H1 (above medium with 1 mg/l selectionagent, for example an herbicide). Carbenicillin and Timentin areantibiotics used to kill the Agrobacterium. The selection agent is usedto allow the growth of the transformed cells.

Callus samples from independent events are tested by PCR. All thesamples tested are positive for the presence of the transformed gene,whereas the non-transformed controls are negative. Callus samples areconfirmed to express the appropriate protein as determined by ELISA.

Callused hypocotyl segments are then placed on B3Z1H1 (MS medium, 3 mg/lbenzylamino purine, 1 mg/l Zeatin, 0.5 gm/l MES [2-(N-morpholino) ethanesulfonic acid], 5 mg/l silver nitrate, 1 mg/l selection agent,Carbenicillin and Timentin) shoot regeneration medium. After shootsstart to regenerate (approximately 3 weeks), hypocotyl segments alongwith the shoots are transferred to B3Z1H3 medium (MS medium, 3 mg/lbenzylamino purine, 1 mg/l Zeatin, 0.5 gm/1 MES [2-(N-morpholino) ethanesulfonic acid], 5 mg/l silver nitrate, 3 mg/l selection agent,Carbenicillin and Timentin) for 3 weeks.

Shoots are excised from the hypocotyl segments and transferred to shootelongation medium MESH10 (MS, 0.5 gm/l MES, 10 mg/l selection agent,Carbenicillin, Timentin) for 2-4 weeks. The elongated shoots arecultured for root induction on MSI.1 (MS with 0.1 mg/l Indolebutyricacid). Once the plants have a well-established root system, these aretransplanted into soil. The plants are acclimated under controlledenvironmental conditions in the Conviron for 1-2 weeks before transferto the greenhouse. The transformed T0 plants self-pollinate in thegreenhouse to obtain T1 seed. Transgenic plants are selected at the T1generation based on resistance to a selection agent. T2 seed (from T1plants) is harvested and sown in soil. T2 plants are grown to maturity,allowed to self-fertilize and set seed. T3 seed (from the T2 plants) isharvested in bulk for each line. Seed oil, protein, digestible protein,and fiber values are measured as discussed in Example 1.

It is claimed:
 1. A transgenic plant, comprising a plant transformationvector comprising a nucleotide sequence that encodes or is thecomplement of a sequence that encodes an IMQ polypeptide comprising anamino acid sequence at least 90% identical to the amino acid sequence asset forth in SEQ ID NO: 94, whereby the transgenic plant has an improvedmeal quality phenotype, relative to control plants.
 2. The transgenicplant of claim 1, wherein the IMQ polypeptide comprises an amino acidsequence at least 95% identical to the amino acid sequence as set forthin SEQ ID NO:
 94. 3. The transgenic plant of claim 1, which is selectedfrom the group consisting of plants of the Brassica species, includingcanola and rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oilpalm, coconut palm, flax, castor, peanut, wheat, oat, and rice.
 4. Thetransgenic plant of claim 1, wherein an improved meal quality phenotypecomprises an increase in available metabolizable energy in meal producedfrom seeds of the transgenic plant, relative to control plants.
 5. Thetransgenic plant of claim 4, wherein an increase in availablemetabolizable energy comprises an altered protein and/or fiber contentin the seeds of the transgenic plant.
 6. The transgenic plant of claim5, wherein the protein content is increased and/or the fiber content isdecreased.
 7. The transgenic plant of claim 4, wherein an increase inavailable metabolizable energy comprises a decreased fiber content inthe seeds of the transgenic plant.
 8. A plant part obtained from theplant according to claim
 1. 9. The plant part of claim 8, which is atransgenic seed, wherein the transgenic seed comprises the planttransformation vector.
 10. Meal, feed, or food produced from thetransgenic seed of claim
 9. 11. A method of producing meal, comprisinggrowing the transgenic plant of claim 1, and recovering meal from theplant, thereby producing meal.
 12. The method of claim 11, wherein themeal is produced from seeds of the plant.
 13. A method of producing animproved meal quality phenotype in a plant, said method comprising: a)introducing into progenitor cells of the plant a plant transformationvector comprising a nucleotide sequence that encodes or is thecompliment of a sequence that encodes an IMQ polypeptide comprising anamino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO: 94, and b) growing the transformed progenitorcells to produce a transgenic plant, wherein the nucleotide sequence isexpressed, and the transgenic plant exhibits an improved meal qualityphenotype relative to control plants, thereby producing the improvedmeal quality phenotype in the plant.
 14. The method of claim 13, whereinthe IMQ polypeptide comprises an amino acid sequence at least 95%identical to the amino acid sequence as set forth in SEQ ID NO:
 94. 15.A plant obtained by a method of claim
 13. 16. The plant of claim 15,which is selected from the group consisting of plants of the Brassicaspecies, including canola and rapeseed, soy, corn, sunflower, cotton,cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, wheat,oat, and rice.
 17. The plant of claim 15, wherein the plant is selectedfrom the group consisting of a plant grown from said progenitor cells, aplant that is the direct progeny of a plant grown from said progenitorcells, and a plant that is the indirect progeny of a plant grown fromsaid progenitor cells.
 18. A method of generating a plant having animproved meal quality phenotype comprising identifying a plant that hasan allele in its ortholog of the A. thaliana IMQ gene where the wildtypeA. thaliana gene has the nucleic acid sequence set forth as SEQ ID NO:93, which allele results in improved meal quality phenotype, compared toplants lacking the allele, wherein identifying a plant comprisesanalyzing the sequence of the allele of the ortholog of the IMQ gene inthe plant; and generating progeny of said identified plant, wherein thegenerated progeny inherit the allele and have the improved meal qualityphenotype, thereby generating a plant having an improved meal qualityphenotype.
 19. The method of claim 18 that employs candidate gene/QTLmethodology.
 20. The method of claim 18 that employs TILLINGmethodology.